Goat anti mouse igg antibody

Total protein was extracted from cells using Tissue or Cell Total Protein Extraction Kit (Sangon Biotech). Equivalent protein from different samples was separated by protein electrophoresis, following by transformation onto PVDF membranes (Merck Millipore, Billerica, MA, USA). The membranes were incubated with the anti-rabbit caspase-3, cleaved-caspase-3, caspase-9, cleaved-caspase-9, PINK-1, Parkin, BNIP3, Beclin-1 and LC3A/B (1:1000, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight after immersed into sealed liquid. After the membranes were washed with TBST for several times, goat anti-mouse IgG antibody (1:1000, Cell Signaling Technology) labeled with horseradish peroxidase were incubated with the membranes as a secondary antibody. Anti-mouse β-actin antibody (1:1000, Cell Signaling Technology) was used as a reference protein for normalization. The gray levels of the protein bands were examined by Image J software.

Cells were harvested and washed twice in cold PBS and lysed in NP40 lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.5% NP40, pH 7.5) supplemented with protease inhibitors (complete Mini EDTA-free protease inhibitor mixture tablets; Roche, Germany) for 30 min on ice and centrifuged at 4°C at 12,000 ×g for 10 min to remove debris. Total proteins in supernatants were quantified with a BCA kit (Thermo Fisher Scientific, USA). Samples were added with loading buffer (consists of β-mercaptoethanol and SDS), denatured at 99°C for 10 min and equilibrated to room temperature, and then 50 or 100 μg of proteins were subjected to 12.5% SDS-PAGE and transferred to a nitrocellulose blotting membrane with a Pyxis machine (GE Healthcare, USA). Rabbit anti-LRRC25ic polyclonal antibody (1 μg/mL) was used to detect LRRC25, β-actin was detected with a mouse anti-human β-actin (1:3,000 dilution, Clone AC-74, Sigma, USA) as a loading control, and secondary antibodies were from Cell Signaling Technology (USA), including HRP-conjugated goat anti-rabbit IgG antibody and goat anti-mouse IgG antibody (1:5,000 dilution). Signals were detected by enhanced chemiluminescence (ECL) Western blot detection reagents (GE, Healthcare) on an ImageQuant^TM LAS500 (GE Healthcare, USA).

Cell lysates were prepared as previously described1 (link). After the bicinchoninic acid (BCA) assay, supernatants containing 25 μg of protein were subjected to 10% SDS/PAGE and transferred onto PVDF membrane (BioRad, CA, USA). After blocking, membranes were incubated with primary antibodies with rabbit or mouse IgG raised against PTEN, phospho-Smad2(Ser-465/467)/3 (Ser-423/425), SMAD2/3 (D7G7), phospho-Akt (Ser473), Akt (C67E7), phospho-ERK1/2 (Thr202/Tyr204), ERK1/2 (3A7), TGF-β, β-Actin, and the secondary antibody was horseradish peroxidase-(HRP-) conjugated goat anti-mouse IgG antibody raised against rabbit or mouse IgG. All antibodies were purchased from Cell Signaling (Danvers, MA, USA) except SMAD7 from Santa Cruz, CA, USA. Blots were developed by detection using ECL substrate (Pierce, Rockford, IL, USA). The Image J software was used to evaluate the band intensity.