Human/Mouse/Rat Phospho-GSK-3 alpha/ beta (S21/S9) Antibody

Detection of Rat GSK-3 alpha/beta by Western Blot

LY294002 inhibits the HYS-32-induced phosphorylation of GSK3 beta-pS9 and GSK3 beta-pY216.(A) Control astrocytes (Con) or astrocytes treated for 24 h with 5 μM HYS-32 (HYS), co-treated for 24 h with 5 μM HYS-32 and 20 μM LY294002 (HYS+LY), or treated with 20 μM LY294002 (LY) were subjected to 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GSK3 beta-pSer9, GSK3 beta-pY216, totalGSK3 beta, or GAPDH. (B) Densitometric analyses of GSK3 beta-pSer9 and GSK3 beta-pY216 expressed as the density of the bands in the treated groups relative to the control. (C) According to the data in (B), the stacked bar graph showing relative percentage ofphospho-GSK3 beta in various treatment. (D)Astrocytes treated as in (A) were fixed in cold acetone and triple-stained for beta-tubulin, N-cadherin, and F-actin. Quantitative analysis of the straight distance between microtubule tips and cell border were performed as described in Materials and Methods. The results were collected from three independent experiments.*p<0.01 compared to HYS using one-way ANOVA with Dunnett’s post-hoctest. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0126217), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Phospho-GSK-3 alpha/ beta (S21/S9) by Western Blot

Galectin-3 expression induces activation of PYK2, STAT1 and GSK3 alpha/ beta signalling. Expression of 37 protein kinases in SW620 cells in response to 10 µg/ml galectin-3 or BSA for 0.5 h was assessed by Proteome Profiler Human Phospho-Kinase Array (A, Percentage changes of the kinases in cell response to galectin-3 in comparison to control are shown at the bottom panel). The presence of galectin-3 increases the phosphorylation of PYK2, GSK3 alpha/ beta, and STAT1 and decreases phosphorylation of STAT3. SW620 cells treated with 10 µg/ml galectin-3 for different times were assessed by immunoblotting using antibodies against p-PYK2, p-STAT-1, p-GSK3 alpha/ beta or p-STAT-3 (B). The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3 alpha/ beta or STAT-3. The band density was quantified and expressed as percentages of phospho-/non-phosphorylated proteins (C). In D and E, SW620 cells were treated with 10 µg/ml galectin-3 or BSA followed by introduction of GSK3 alpha/ beta inhibitor SB 216763 (SB) or PKY2 inhibitor PF-431396 (PF) for 15 min and the levels of phosphorylated PYK2, STAT-1, GSK3 alpha/ beta or STAT-3 were analysed by immunoblotting. The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3 alpha/ beta or STAT-3. The densities of the blots from three independent experiments were quantified and are expressed as the percentage of phosphorylated/non-phosphorylated levels of each protein. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37055381), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Phospho-GSK-3 alpha/ beta (S21/S9) by Western Blot

Galectin-3 expression induces activation of PYK2, STAT1 and GSK3 alpha/ beta signalling. Expression of 37 protein kinases in SW620 cells in response to 10 µg/ml galectin-3 or BSA for 0.5 h was assessed by Proteome Profiler Human Phospho-Kinase Array (A, Percentage changes of the kinases in cell response to galectin-3 in comparison to control are shown at the bottom panel). The presence of galectin-3 increases the phosphorylation of PYK2, GSK3 alpha/ beta, and STAT1 and decreases phosphorylation of STAT3. SW620 cells treated with 10 µg/ml galectin-3 for different times were assessed by immunoblotting using antibodies against p-PYK2, p-STAT-1, p-GSK3 alpha/ beta or p-STAT-3 (B). The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3 alpha/ beta or STAT-3. The band density was quantified and expressed as percentages of phospho-/non-phosphorylated proteins (C). In D and E, SW620 cells were treated with 10 µg/ml galectin-3 or BSA followed by introduction of GSK3 alpha/ beta inhibitor SB 216763 (SB) or PKY2 inhibitor PF-431396 (PF) for 15 min and the levels of phosphorylated PYK2, STAT-1, GSK3 alpha/ beta or STAT-3 were analysed by immunoblotting. The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3 alpha/ beta or STAT-3. The densities of the blots from three independent experiments were quantified and are expressed as the percentage of phosphorylated/non-phosphorylated levels of each protein. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37055381), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Phospho-GSK-3 alpha/ beta (S21/S9) by Western Blot

Galectin-3 expression induces activation of PYK2, STAT1 and GSK3 alpha/ beta signalling. Expression of 37 protein kinases in SW620 cells in response to 10 µg/ml galectin-3 or BSA for 0.5 h was assessed by Proteome Profiler Human Phospho-Kinase Array (A, Percentage changes of the kinases in cell response to galectin-3 in comparison to control are shown at the bottom panel). The presence of galectin-3 increases the phosphorylation of PYK2, GSK3 alpha/ beta, and STAT1 and decreases phosphorylation of STAT3. SW620 cells treated with 10 µg/ml galectin-3 for different times were assessed by immunoblotting using antibodies against p-PYK2, p-STAT-1, p-GSK3 alpha/ beta or p-STAT-3 (B). The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3 alpha/ beta or STAT-3. The band density was quantified and expressed as percentages of phospho-/non-phosphorylated proteins (C). In D and E, SW620 cells were treated with 10 µg/ml galectin-3 or BSA followed by introduction of GSK3 alpha/ beta inhibitor SB 216763 (SB) or PKY2 inhibitor PF-431396 (PF) for 15 min and the levels of phosphorylated PYK2, STAT-1, GSK3 alpha/ beta or STAT-3 were analysed by immunoblotting. The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3 alpha/ beta or STAT-3. The densities of the blots from three independent experiments were quantified and are expressed as the percentage of phosphorylated/non-phosphorylated levels of each protein. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37055381), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Phospho-GSK-3 alpha/ beta (S21/S9) by Western Blot

Galectin-3 expression induces activation of PYK2, STAT1 and GSK3 alpha/ beta signalling. Expression of 37 protein kinases in SW620 cells in response to 10 µg/ml galectin-3 or BSA for 0.5 h was assessed by Proteome Profiler Human Phospho-Kinase Array (A, Percentage changes of the kinases in cell response to galectin-3 in comparison to control are shown at the bottom panel). The presence of galectin-3 increases the phosphorylation of PYK2, GSK3 alpha/ beta, and STAT1 and decreases phosphorylation of STAT3. SW620 cells treated with 10 µg/ml galectin-3 for different times were assessed by immunoblotting using antibodies against p-PYK2, p-STAT-1, p-GSK3 alpha/ beta or p-STAT-3 (B). The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3 alpha/ beta or STAT-3. The band density was quantified and expressed as percentages of phospho-/non-phosphorylated proteins (C). In D and E, SW620 cells were treated with 10 µg/ml galectin-3 or BSA followed by introduction of GSK3 alpha/ beta inhibitor SB 216763 (SB) or PKY2 inhibitor PF-431396 (PF) for 15 min and the levels of phosphorylated PYK2, STAT-1, GSK3 alpha/ beta or STAT-3 were analysed by immunoblotting. The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3 alpha/ beta or STAT-3. The densities of the blots from three independent experiments were quantified and are expressed as the percentage of phosphorylated/non-phosphorylated levels of each protein. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37055381), licensed under a CC-BY license. Not internally tested by R&D Systems.