Rabbit anti β actin primary antibody

Western blotting to determine the expression of nanobodies was carried out using a specific goat anti-alpaca polyclonal antibody conjugated to HRP (Jackson ImmunoResearch), while actin was blotted using a rabbit anti-β-actin primary antibody (Sigma-Aldrich) and then goat anti-rabbit secondary antibody conjugated to HRP. Amersham ECL Western Blotting Detection Reagents (GE Healthcare) or Chemiluminescent HRP Substrate (Millipore) were used for detection. Note that the anti-alpaca polyclonal antibody exhibits varying levels of affinity for different nanobodies; hence, band intensities for different nanobodies in western blots are not directly comparable.

Cell pellets were lysed in lysis buffer (PBS + 1% Triton X100) with cOmplete protease inhibitor (Roche) on ice, briefly sonicated on ice, and centrifuged at maximum speed for 20 min at 4°C. Supernatants containing total lysates were collected on ice and quantified for total protein contents using the BCA assay kit (Thermo Fisher Scientific). Total lysate (10 μg) was loaded on NuPAGE 10% Bis-Tris, 1.5 mm, Protein Gel, 10-well (Thermo Fisher Scientific) and run at 120 V for approximately 90 min for SDS-PAGE. Gels were transferred to a 0.2 μm nitrocellulose membrane (Amersham) at 250 mA for 90 min. DJ-1 was detected with mouse anti-DJ-1 primary antibody (Enzo Life Sciences), and actin was detected with rabbit anti-β-actin primary antibody (Sigma Aldrich), both diluted 1:8,000 in 5% BSA in Tris-buffered saline-Tween 20 (TBST) and incubated overnight at 4°C. Horseradish peroxidase conjugated secondary anti-mouse and anti-rabbit antibodies were diluted 1:10,000 and incubated at room temperature for 1 h. Signals were detected using Pierce ECL plus detection reagent (Thermo Fisher Scientific) and read on ChemiDoc (Bio-Rad). Band intensities were calculated using ImageJ (NIH) and Image Lab (Bio-Rad) software. Data from a minimum of three independent experiments were analyed statistically using t tests.

Western blot analysis was applied to determine the levels of protein expression in cells according to previous studies^{25 (link)–28 (link)}. Briefly, the proteins in the four groups were extracted by RIPA buffer (Beyotime, China) and transfected onto the PVDF membranes, then the PVDF membranes were incubated with 1:500 diluted mouse anti-p38 primary antibody (Abcam, UK), 1:500 diluted mouse anti-p-p38 primary antibody (Santa Cruz, USA), 1:500 diluted mouse anti-α-SM-actin primary antibody (Abcam, UK) and 1:1000 diluted rabbit anti-β-actin primary antibody (Sigma-Aldrich Co., USA) overnight at 4 °C. The following day, the membranes were probed with the 1:5000 IRDye diluted 700-conjugated affinity-purified goat anti-mouse second antibody (Rockland Immunochemicals, USA) or 1:5000 diluted IRDye 800-conjugated affinity-purified goat anti-rabbit second antibody (1:5000, Rockland Immunochemicals, USA) for 60 min at room temperature. Then protein bands were visualized using Odyssey laser scanning system (LI-COR Inc., USA) and the intensities were quantified by Odyssey 3.0 image analysis system software.