Anti hspa8

The antibodies used for immunoblot, immunoprecipitation, immunofluorescence or in vitro uptake assays were listed as following: anti-LAMP2A (125,068), anti-MARCHF5 (185,054), anti-HSPD1 (13,532), anti-SQSTM1 (109,012), anti-MFN1 (57,602), anti-MFN2 (56,889), anti-HSPA8 (51,052), anti-DNM1L (184,247), anti-RNASE/RNaseA (94,417) were purchased from Abcam; anti-ACTB (AC037) from ABclonal; anti-COX4I1 (11,242-1-AP), anti-Flag (20,543-1-AP), anti-MYC (60,003-2-Ig), anti-MIEF2 (16,413-1-AP) from Proteintech; anti-TH (T1299) from Sigma-Aldrich; anti-VDAC1 (4661) from Cell Signaling Technology; HRP-labeled goat anti-mouse IgG (AS003) and goat anti-rabbit IgG (AS014) from ABclonal; Alexa Fluor 488-adsorbed goat anti-rabbit IgG (A11034), Alexa Fluor 568-adsorbed goat anti-rabbit IgG (A11036) and Alexa Fluor 647-adsorbed goat anti-mouse IgG (A21236) from Invitrogen. The chemicals used were listed as following: ammonium chloride (Sigma-Aldrich, A9434), leupeptin (MP Biomedicals, 03476–89-7), 3-methyladenine (Selleck, S2767).

Cells were lysed in RIPA buffer (Genestar) supplemented with protease inhibitor cocktail (MCE) and proteins were extracted after centrifugation of the supernatant at 12,000×g and 4 °C. Proteins were loaded into an electrophoresis chamber for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred to nitrocellulose membranes. Membranes were blocked in TBST containing 5% skim milk powder for 1 h and were then incubated with the following primary antibodies overnight at 4 °C, anti-HSPA8 (Abcam, Cat. # ab51052); anti-P16 (ABclonal, Cat. # A0262), anti-P21 (ABclonal, Cat. # A19094), anti-P38 (Cell Signaling Technology, Cat. # 9212S), anti-pp38 (Cell Signaling Technology, Cat. # 9211S), LaminB1 (Sino Biological, Cat. # 101237-T32), anti-actin (Sungene Biotech, Cat. # KM9001) and anti-tubulin (Sungene Biotech, Cat. # KM9003T). After washing with TBST, membranes were incubated for 1 h at room temperature with the corresponding secondary antibody. After washing with TBST three times, membranes were incubated with ECL solution (Thermo) and chemical signals were acquired using a ChemiDoc XRS + molecular imager (BioRad). The band signals were analyzed with Image Lab software.