Human SSBP1 Antibody

Detection of SSBP1 by Western Blot

Proteasome action is required at late stages of mitophagy. (A) Scheme of treatment time points for proteasome inhibition experiments. Bold text indicates treatment with CCCP while normal text indicates treatment with proteasome inhibitor MG132. (B and C) Western blot analysis of proteasome influence on the indicated inner mitochondrial proteins. (D) Representative images of WT-parkin HeLa cells showing SSBP1 (magenta) and TOM20 (green) after 24 h of CCCP treatment or CCCP treatment combined with MG132 added after 8 h of mitochondria depolarization. Scale bar = 5 µm. (E and F) Single cell analysis of (E) mean number of SSSP1 clumps and (F) relative average SSBP1 clump area per cell. Bars: Mean ± SEM. ns: non-significant; *: p≤0.05, ***: p≤0.001. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/40396016), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of SSBP1 by Immunohistochemistry

Parkin activity is essential for the removal of depolarized mitochondria.A, immunofluorescence staining of WT-parkin and C431A-parkin cells after early (2 h) and late (18 h) depolarization times. Scale bars represent 5 μm. B, experimental setup combining subcellular protein fractionation with peptide dimethyl labeling and high pH reversed-phase fractionation during “early” and “late” mitophagy stages in WT-parkin and C431A-parkin cells. Basic workflow applied for proteome and phosphoproteome analysis. C, correlation plot between replicates after log2 transformation of protein ratios. Common light dimethyl labeling channel (0 h postdepolarization) allows comparison between “early” and “late” triple dimethyl-labeling sets per cell line. Parkin-dependent degradation of mitochondrial proteins reflected by shifting data points (density colored points) from “unregulated” toward “downregulated” in WT-parkin expressing cells in comparison to total proteome (gray points). Number of valid pairs per condition and Spearman rank correlation (in brackets) indicated for total (T) and mitochondrial annotated proteins (M). Image collected and cropped by CiteAb from the following open publication (https://linkinghub.elsevier.com/retrieve/pii/S1535947621001638), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of SSBP1 by Immunohistochemistry

Autophagy activation and delivery to lysosomes of inner- mitochondrial material. (A and B) Influence of BafA inhibition on TOM20-negative mitochondrial-aggresomes. (A) Representative immunofluorescence images showing TOM20 (green) and SSBP1 (magenta) structures at the indicated CCCP-treatment time points in combination with autophagy inhibition (BafA). (B) Single-cell analysis of mean number of mitochondrial SSBP1 and TOM20 puncta in (A). (C) COX IV-positive puncta (magenta) are positive for EGFP-LC3B (yellow). Arrows indicate COX IV puncta partially engaged with EGFP-LC3B structures. Closed arrowheads indicate complete overlap of COX IV puncta and EGFP-LC3B. Open arrowheads indicate non co-localizing COX IV puncta with EGFP-LC3B. (D) Representative immunofluorescence images showing COX IV-positive puncta (green) co-localizing with LysoTracker (magenta) at the indicated time points. Arrows indicate mitochondrial puncta while arrowheads indicate inner mitochondrial aggregates. (E and F) Representative immunofluorescence images showing (E) COX IV-positive (magenta) or (F) SSBP1-positive mitochondrial aggregates (magenta) close to LAMP1 patches (green) at the indicated time points. Arrows indicate mitochondrial puncta while arrowheads indicate inner mitochondrial aggregates. ****: p ≤0.0001. All scale bars = 5 µm. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/40396016), licensed under a CC-BY license. Not internally tested by R&D Systems.