Detection of Mouse p62/SQSTM1 by Simple WesternTM.
Simple Western lane view shows lysates of RAW 264.7 mouse monocyte/macrophage cell line untreated (-) or treated (+) with 1 µg/mL LPS for 24 hours, loaded at 0.2 mg/mL. A specific band was detected for p62/SQSTM1 at approximately 66 kDa (as indicated) using 20 µg/mL of Mouse Anti-Human/Mouse/Rat p62/SQSTM1 Monoclonal Antibody (Catalog # MAB8028). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.
p62/SQSTM1 Specificity is Shown by Immunocytochemistry in Knockout Cell Line.
p62/SQSTM1 was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line but is not detected in p62/SQSTM1 knockout (KO) HeLa cell line using Mouse Anti-Human/Mouse/Rat p62/SQSTM1 Monoclonal Antibody (Catalog # MAB8028) at 3 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red;
NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.
Western Blot Shows Human p62/SQSTM1 Specificity Using Knockout Cell Line.
Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and p62/SQSTM1 knockout HeLa cell line (KO). PVDF membrane was probed with 2 µg/mL of Mouse Anti-Human/Mouse/Rat p62/SQSTM1 Monoclonal Antibody (Catalog # MAB8028) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (
HAF018). A specific band was detected for p62/SQSTM1 at approximately 62 kDa (as indicated) in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. GAPDH (
MAB5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Detection of Human p62/SQSTM1 by Western Blot
The combination of BRZ and TMZ increased cell death in GBM stem-like cells by activating autophagy (A,B) Autophagy marker LC3 immunostaining of U251_Ctrl and U251_CA2 cells after TMZ and ACZ/BRZ stimulation for 24 h. Co-treatment with TMZ and BRZ induced the expression of LC3 puncta in U251_CA2 cells (scale bar: 50 μm). (C,D) Western Blotting of autophagy-related proteins and CA2 protein in U251_Ctrl and U251_CA2 cells with the same treatment as in (A). TMZ plus BRZ did not increase the protein expression of LC3II in U251_Ctrl cells compared to TMZ treatment alone (C) but increased in U251_CA2 cells (D) (n = 3). (E–G) Western Blotting of autophagy-related proteins and CA2 protein in GBM stem cells with the same treatment as in Figure 5E–G for 24 h stimulation. Compared with the control group, TMZ monotherapy has a tendency to increase the protein level of LC3II, but the expression of it is significantly increased in BRZ alone and TMZ combined with BRZ treatment (n = 3). Results were obtained from three independent experiments. Data are presented as mean ± SEM, One-way ANOVA was used to analyze the data, * p < 0.05; ** p < 0.01; *** p < 0.001, ns: not significant. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35008590), licensed under a CC-BY license. Not internally tested by R&D Systems.
Western Blot Shows Human p62/SQSTM1 Specificity Using Knockout Cell Line.
Western blot shows lysates of U2OS human osteosarcoma cell line and p62/SQSTM1 knockout U2OS cell line (KO). Nitrocellulose membrane was probed with 0.5 µg/mL of Mouse Anti-Human/Mouse/Rat p62/SQSTM1 Monoclonal Antibody (Catalog # MAB8028) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody. A specific band was detected for p62/SQSTM1 at approximately 62 kDa (as indicated) in the parental U2OS cell line, but is not detectable in knockout U2OS cell line. The Ponceau stained transfer of the blot is shown. This experiment was conducted under reducing conditions. Image, protocol, and testing courtesy of YCharOS Inc. See ycharos.com for additional details.
Detection of SQSTM1/p62 by Immunoprecipitation
Immunoprecipitation was performed on cell lysate of U2OS human osteosarcoma cell line using 1.0 μg of Mouse Anti-Human SQSTM1/p62 Monoclonal Antibody (Catalog # MAB8028) pre-coupled to protein G or protein A beads. Immunoprecipitated SQSTM1/p62 was detected with Rabbit Anti-SQSTM1/p62 Monoclonal Antibody (Catalog # MAB80281). The Ponceau stained transfers of each blot are shown. SM=10% starting material; UB=10% unbound fraction; IP=immunoprecipitated. Image, protocol, and testing courtesy of YCharOS Inc. (ycharos.com).
Detection of Human p62/SQSTM1 by Western Blot
The combination of BRZ and TMZ increased cell death in GBM stem-like cells by activating autophagy (A,B) Autophagy marker LC3 immunostaining of U251_Ctrl and U251_CA2 cells after TMZ and ACZ/BRZ stimulation for 24 h. Co-treatment with TMZ and BRZ induced the expression of LC3 puncta in U251_CA2 cells (scale bar: 50 μm). (C,D) Western Blotting of autophagy-related proteins and CA2 protein in U251_Ctrl and U251_CA2 cells with the same treatment as in (A). TMZ plus BRZ did not increase the protein expression of LC3II in U251_Ctrl cells compared to TMZ treatment alone (C) but increased in U251_CA2 cells (D) (n = 3). (E–G) Western Blotting of autophagy-related proteins and CA2 protein in GBM stem cells with the same treatment as in Figure 5E–G for 24 h stimulation. Compared with the control group, TMZ monotherapy has a tendency to increase the protein level of LC3II, but the expression of it is significantly increased in BRZ alone and TMZ combined with BRZ treatment (n = 3). Results were obtained from three independent experiments. Data are presented as mean ± SEM, One-way ANOVA was used to analyze the data, * p < 0.05; ** p < 0.01; *** p < 0.001, ns: not significant. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35008590), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human p62/SQSTM1 by Western Blot
The combination of BRZ and TMZ increased cell death in GBM stem-like cells by activating autophagy (A,B) Autophagy marker LC3 immunostaining of U251_Ctrl and U251_CA2 cells after TMZ and ACZ/BRZ stimulation for 24 h. Co-treatment with TMZ and BRZ induced the expression of LC3 puncta in U251_CA2 cells (scale bar: 50 μm). (C,D) Western Blotting of autophagy-related proteins and CA2 protein in U251_Ctrl and U251_CA2 cells with the same treatment as in (A). TMZ plus BRZ did not increase the protein expression of LC3II in U251_Ctrl cells compared to TMZ treatment alone (C) but increased in U251_CA2 cells (D) (n = 3). (E–G) Western Blotting of autophagy-related proteins and CA2 protein in GBM stem cells with the same treatment as in Figure 5E–G for 24 h stimulation. Compared with the control group, TMZ monotherapy has a tendency to increase the protein level of LC3II, but the expression of it is significantly increased in BRZ alone and TMZ combined with BRZ treatment (n = 3). Results were obtained from three independent experiments. Data are presented as mean ± SEM, One-way ANOVA was used to analyze the data, * p < 0.05; ** p < 0.01; *** p < 0.001, ns: not significant. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35008590), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human p62/SQSTM1 by Western Blot
The combination of BRZ and TMZ increased cell death in GBM stem-like cells by activating autophagy (A,B) Autophagy marker LC3 immunostaining of U251_Ctrl and U251_CA2 cells after TMZ and ACZ/BRZ stimulation for 24 h. Co-treatment with TMZ and BRZ induced the expression of LC3 puncta in U251_CA2 cells (scale bar: 50 μm). (C,D) Western Blotting of autophagy-related proteins and CA2 protein in U251_Ctrl and U251_CA2 cells with the same treatment as in (A). TMZ plus BRZ did not increase the protein expression of LC3II in U251_Ctrl cells compared to TMZ treatment alone (C) but increased in U251_CA2 cells (D) (n = 3). (E–G) Western Blotting of autophagy-related proteins and CA2 protein in GBM stem cells with the same treatment as in Figure 5E–G for 24 h stimulation. Compared with the control group, TMZ monotherapy has a tendency to increase the protein level of LC3II, but the expression of it is significantly increased in BRZ alone and TMZ combined with BRZ treatment (n = 3). Results were obtained from three independent experiments. Data are presented as mean ± SEM, One-way ANOVA was used to analyze the data, * p < 0.05; ** p < 0.01; *** p < 0.001, ns: not significant. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35008590), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human p62/SQSTM1 by Western Blot
The combination of BRZ and TMZ increased cell death in GBM stem-like cells by activating autophagy (A,B) Autophagy marker LC3 immunostaining of U251_Ctrl and U251_CA2 cells after TMZ and ACZ/BRZ stimulation for 24 h. Co-treatment with TMZ and BRZ induced the expression of LC3 puncta in U251_CA2 cells (scale bar: 50 μm). (C,D) Western Blotting of autophagy-related proteins and CA2 protein in U251_Ctrl and U251_CA2 cells with the same treatment as in (A). TMZ plus BRZ did not increase the protein expression of LC3II in U251_Ctrl cells compared to TMZ treatment alone (C) but increased in U251_CA2 cells (D) (n = 3). (E–G) Western Blotting of autophagy-related proteins and CA2 protein in GBM stem cells with the same treatment as in Figure 5E–G for 24 h stimulation. Compared with the control group, TMZ monotherapy has a tendency to increase the protein level of LC3II, but the expression of it is significantly increased in BRZ alone and TMZ combined with BRZ treatment (n = 3). Results were obtained from three independent experiments. Data are presented as mean ± SEM, One-way ANOVA was used to analyze the data, * p < 0.05; ** p < 0.01; *** p < 0.001, ns: not significant. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35008590), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of p62/SQSTM1 by Western Blot
CSE induces autophagy in ASMCs. (A) Representative Western blots and image analysis of CSE-induced p62, beclin-1, and LC3A/B protein expression. GAPDH served as reference protein and p < 0.05 was considered significant. (B) Representative confocal microscopy on mitochondria-LC3 co-localization (white arrow) after CSE-stimulation (60×, green: cytochrome C, red: LC3A/B, blue: DAPI). Statistics were calculated by Mann–Whitney U-test. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36430467), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of p62/SQSTM1 by Western Blot
MDP reduces intestinal barrier damage and activates autophagy in vitro. (A) Schematic diagram of the administration of MDP (0, 1, 5, 10 μg/ml, respectively) in Caco-2 cells. (B) Western blotting analysis for NOD2, LC3 І, and LC3 ІІ with GAPDH as the internal standard protein in Caco-2 cells. Representative images of the immune blotting were shown. (C,D) Quantification of the relative expression of NOD2 and LC3 ІІ in panel (B) by ImageJ, respectively. Results were from three independent experiments. (E) Cytotoxicity of Caco-2 cells with the administration of with LPS (0, 1, 10, 100 μg/mL, respectively) for 24 h by testing LDH OD value. (F) Relative RNA levels of IL-1 beta of Caco-2 cells with the administration of LPS. (G) Western blotting analysis for ZO-1, p62, and LC3 ІІ with GAPDH as the internal standard protein in Caco-2 cells of the groups (Control, MDP, LPS, and LPS + MDP), respectively. Representative images of three duplicate samples of the immune blotting were shown. (H–J) Quantification of the relative expression of ZO-1, p62, and LC3 ІІ in panel (G) by ImageJ, respectively. (K) Cytotoxicity of Caco-2 cells of the groups (Control, MDP, LPS, and LPS + MDP) by testing LDH OD value. Data were expressed as mean ± SEM. *p <0.05, **p <0.01, ns, not significant, One-way ANOVA. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36506547), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of p62/SQSTM1 by Western Blot
MDP activates autophagy in colon of DSS-induced mice. (A) Western blotting analysis for p62, LC3 І, and LC3 ІІ with beta-Actin as the internal standard protein in the colon of PBS, MDP, DSS + PBS, and DSS + MDP group mice. Representative images of three duplicate samples of the immune blotting were shown. (B–D) Quantification of the relative expression of p62, LC3 І and LC3 ІІ in panel (A) by ImageJ, respectively. Data were expressed as mean ± SEM. *p <0.05, **p <0.01, ns, not significant, One-way ANOVA. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36506547), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of p62/SQSTM1 by Western Blot
MDP activates autophagy in colon of DSS-induced mice. (A) Western blotting analysis for p62, LC3 І, and LC3 ІІ with beta-Actin as the internal standard protein in the colon of PBS, MDP, DSS + PBS, and DSS + MDP group mice. Representative images of three duplicate samples of the immune blotting were shown. (B–D) Quantification of the relative expression of p62, LC3 І and LC3 ІІ in panel (A) by ImageJ, respectively. Data were expressed as mean ± SEM. *p <0.05, **p <0.01, ns, not significant, One-way ANOVA. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36506547), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of p62/SQSTM1 by Western Blot
CSE modulates mitochondrial homeostasis by mitophagy and lysosome activity. (A) Representative Western blots and image analysis of p62, beclin-1, and LC3A/B protein of control and COPD ASMCs; GAPDH served as reference protein; p < 0.05 was considered significant. Statistics were calculated by Student’s t-test. (B) Representative confocal microscopy of co-localization mitochondria-LC3 (60×, green: cytochrome C, red: LC3A/B, blue: DAPI, white arrow shows co-localization). (C) Representative Western blots and image analysis of EEA1 and LAMP-1 protein of control and COPD ASMCs; GAPDH was used as reference protein, p < 0.05 was considered significant. Statistics were calculated by Student’s t-test. (D), Representative confocal microscopy of mitochondria–lysosome co-localization (60×, red: TOMM20, green: LAMP1, blue: DAPI, white arrow shows co-localization. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36430467), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of p62/SQSTM1 by Western Blot
CSE modulates mitochondrial homeostasis by mitophagy and lysosome activity. (A) Representative Western blots and image analysis of p62, beclin-1, and LC3A/B protein of control and COPD ASMCs; GAPDH served as reference protein; p < 0.05 was considered significant. Statistics were calculated by Student’s t-test. (B) Representative confocal microscopy of co-localization mitochondria-LC3 (60×, green: cytochrome C, red: LC3A/B, blue: DAPI, white arrow shows co-localization). (C) Representative Western blots and image analysis of EEA1 and LAMP-1 protein of control and COPD ASMCs; GAPDH was used as reference protein, p < 0.05 was considered significant. Statistics were calculated by Student’s t-test. (D), Representative confocal microscopy of mitochondria–lysosome co-localization (60×, red: TOMM20, green: LAMP1, blue: DAPI, white arrow shows co-localization. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36430467), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of p62/SQSTM1 by Western Blot
MDP reduces intestinal barrier damage and activates autophagy in vitro. (A) Schematic diagram of the administration of MDP (0, 1, 5, 10 μg/ml, respectively) in Caco-2 cells. (B) Western blotting analysis for NOD2, LC3 І, and LC3 ІІ with GAPDH as the internal standard protein in Caco-2 cells. Representative images of the immune blotting were shown. (C,D) Quantification of the relative expression of NOD2 and LC3 ІІ in panel (B) by ImageJ, respectively. Results were from three independent experiments. (E) Cytotoxicity of Caco-2 cells with the administration of with LPS (0, 1, 10, 100 μg/mL, respectively) for 24 h by testing LDH OD value. (F) Relative RNA levels of IL-1 beta of Caco-2 cells with the administration of LPS. (G) Western blotting analysis for ZO-1, p62, and LC3 ІІ with GAPDH as the internal standard protein in Caco-2 cells of the groups (Control, MDP, LPS, and LPS + MDP), respectively. Representative images of three duplicate samples of the immune blotting were shown. (H–J) Quantification of the relative expression of ZO-1, p62, and LC3 ІІ in panel (G) by ImageJ, respectively. (K) Cytotoxicity of Caco-2 cells of the groups (Control, MDP, LPS, and LPS + MDP) by testing LDH OD value. Data were expressed as mean ± SEM. *p <0.05, **p <0.01, ns, not significant, One-way ANOVA. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36506547), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of p62/SQSTM1 by Western Blot
CSE induces autophagy in ASMCs. (A) Representative Western blots and image analysis of CSE-induced p62, beclin-1, and LC3A/B protein expression. GAPDH served as reference protein and p < 0.05 was considered significant. (B) Representative confocal microscopy on mitochondria-LC3 co-localization (white arrow) after CSE-stimulation (60×, green: cytochrome C, red: LC3A/B, blue: DAPI). Statistics were calculated by Mann–Whitney U-test. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36430467), licensed under a CC-BY license. Not internally tested by R&D Systems.
