Goat anti mouse igg antibody

The protein extraction and Western blot analysis were performed as previous described.²² The antibody information was as following: kindlin‐2 antibody (13562s, Cell Signaling Technology), Bax antibody (14796, Cell Signaling Technology), Bcl‐2 antibody (ab194583, Abcam), GRP78 antibody (GRP78, 3183, Cell Signaling Technology), CHOP antibody (2895, Cell Signaling Technology), PDI antibody (3501, Cell Signaling Technology), Ero1‐Lα antibody (ab81959, Abcam), Drp‐1 antibody (ab184247, Abcam), Tfam antibody (ab131607, Abcam), ND3 (ab192306, Abcam), Mfn‐2 (9482, Cell Signaling Technology), Fis‐1 (ab71498, Abcam), ATPB (ab170947, Abcam), β actin Antibody (HRP‐60008, Proteintech), Goat anti‐rabbit IgG antibody (SA00001‐2, Proteintech) and Goat anti‐mouse IgG antibody (SA00001‐1, Proteintech). The Image J software was used for quantitative analysing, and relative protein levels were expressed as the intensity ratio of target protein and β actin.

The extraction of total protein was using the RIPA buffer (1% Triton X-100, 50 mM Tris-HCl pH 7.2, 0.1% SDS and 1 mM EDTA, 150 mM NaCl, 1% sodium deoxycholate). Using the bicinchoninic acid method to quantify, 20–25 μg protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, the protein was transferred to 0.45 μM nitrocellulose membranes. Membranes were soaked in TBS-T buffer containing 5% bovine serum albumin for about 1 h and incubated with the following primary antibodies (rabbit anti-PELP1 antibody, A3189, 1:1,000, ABclonal) overnight at 4°C. anti-c-Src antibody (A0324, 1:1,000, ABclonal), anti-Phospho-Src-Y529-antibody (AP0185, 1:500, ABclonal) and mouse polyclonal GAPDH antibody (SC-47724, 1:1,000, Santa Cruz). After washing with TBS-T, the membrane was then conjugated with horseradish peroxidase (HRP) goat anti-rabbit IgG antibody (1:5,000, Proteintech) or goat anti-mouse IgG antibody (1:20,000; Proteintech) at room temperature Hybridization for 1 h. The blots were photographed by the Fujifilm LAS4000 mini luminescent image analyzer, and the density of the protein bands was quantified by Multi Gauge V3.0 analyzer software (FujiFilm Corp., Tokyo, Japan).

Total protein was extracted from lung tissues or 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 SIRT1, TIMP-1, MMP-9, CHOP, GRP78, caspase-12 or caspase-3 antibodies (1:1000 dilution, Proteintech, Wuhan, China) at 4 °C overnight after immersed into blocking buffer. After the membranes were washed with TBST for several times, goat anti-mouse IgG antibody (1:5000, Proteintech) labelled with horseradish peroxidase were incubated with the membranes as a secondary antibody. Anti-mouse β-actin antibody (1:5000, Proteintech) was used as a reference protein for normalization. The grey levels of the protein bands were examined by Image J software.