Article Title: Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy
Figure Lengend Snippet: G-TPP leads to recruitment of autophagy adapters and degradation of mitochondria ( A ) HeLa cells stably expressing untagged Parkin were treated with 10 µM G-TPP for 8 h. Western blots were prepared from cell lysates and probed with antibodies against LC3, phospho-TBK1 (Ser172) and TBK1. GAPDH was used as a loading control. Upon 8 h the levels of LC3-I and LC3-II were both increased. At 8 h after treatment with G-TPP but not at 4 or 24 h, TBK1 was phosphorylated. ( B ) HeLa cells stably expressing EGFP-Parkin were treated with 10 µM G-TPP and fixed 8 h after treatment. Cells were stained with antibodies against the autophagy adapter proteins NBR1, NDP52, OPTN, p62, and TAX1BP1 (red). Mitochondria were counterstained with TOM20 antibodies (cyan), nuclei with Hoechst (blue). EGFP-Parkin epifluorescence is shown in green. Scale bar corresponds to 10 µM. ( C ) HeLa cells stably expressing EGFP-Parkin and the reporter protein mitoKeima were treated with 10 µM CCCP or G-TPP and imaged over time. The ratio of ‘neutral’ mitoKeima to ‘acidic’ mitoKeima was calculated as readout for mitophagy. Parkin translocation was monitored at the same time. Values for Parkin translocation and mitophagy were normalized to 12 h treatment with 10 µM CCCP as positive control and DMSO as negative control (two-way ANOVA with Tukey’s post-hoc test, ** p
Article Snippet: Antibodies The following antibodies have been used for western blot (WB) or immunofluorescence (IF): beta III tubulin (#5568, CST, WB: 1/2,000 or AB9354, Millipore, IF: 1/250), FLAG (F3165, Sigma, WB: 1/150,000), GAPDH (H86504M, Meridian Life Sciences, WB: 1/500,000), LC3B (NB100-2220, Novus Biologicals, WB: 1/5,000), Miro1 (H00055288-M01, Novus Biologicals, WB: 1/500), Mitofusin 1 (ab57602, Abcam, WB: 1/5,000), Mitofusin 2 (ab56889, Abcam, WB: 1/5,000), NBR1 (H00004077-M01, Abnova, WB: 1/500, IF: 1/100), NDP52 (12229-1-AP, PTG, WB: 1/1,000, IF: 1/400), OPTN (sc-166576, Santa Cruz, IF: 1/100), OPTN (10837-1-AP, PTG, WB: 1/5,000), p38 (#9212, CST, WB: 1/2,000), p62 (610832, BD Biosciences, WB: 1/2,000, IF:1/500), Parkin (#4211, CST, WB: 1/3,000), PGAM5 (ab12653, Abcam, WB: 1/5,000), PINK1 (#6946, CST, WB: 1/2,000, IF: 1/1,000), PINK1 (BC100-494, Novus Biologicals, WB: 1/2,000), TAX1BP1 (#5105, CST, WB: 1/2,000, IF: 1/400), TBK1 (#3504, CST, WB: 1/1,000), pS172-TBK1 (#5483, CST, WB: 1/1,000), TOM20 rabbit (11802-1-AP, PTG, IF: 1/2,000), TOM20 mouse (sc-17764, Santa Cruz, IF: 1/100), TOM70 (14528-1-AP, PTG, WB: 1/5,000), TRAP1 (#13405, CST, WB: 1/1,000), ubiquitin (#3933, CST, WB: 1/2,000), pS65-Ub (in-house [ , ], WB: 1/15,000, IF: 1/250), VDAC1 (ab14734, Abcam, WB: 1/10,000), vinculin (V9131, Sigma, WB: 1/500,000).
Techniques: Stable Transfection, Expressing, Western Blot, Staining, Translocation Assay, Positive Control, Negative Control