Goat anti mouse igg hrp secondary antibody

The sequence encoding PARK6 was amplified from the plasmid pcDNA3.1/Pink1 (Meissner et al., 2011 (link)) and cloned into the pcDNA5/mCherry-sfGFP vector (Khmelinskii et al., 2012 (link)). pcDNA5/PARK6-mCherry-sfGFP(cp8) was cloned by overlap assembly PCR of a human codon-optimized sfGFP(cp8), which was obtained by full gene synthesis, into the pcDNA5/PARK6-mCherry-sfGFP background.
HEK293T cells were grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum at 37°C in 5% (vol/vol) CO₂. Transient transfections were performed using 25-kDa linear polyethylenimine (Polysciences, Warrington, PA; Durocher et al., 2002 (link)) and harvested 24 h later. To block mitochondrial protein import, cells were incubated for 3 h with 10 μM carbonyl cyanide m-chlorophenyl hydrazone (Sigma-Aldrich) before cell lysis.
Whole-cell extracts were separated by SDS–PAGE, followed by blotting onto a polyvinylidene fluoride membrane and probed with a monoclonal anti-GFP antibody (11814460001; Roche, Sigma-Aldrich). A secondary goat anti-mouse IgG-HRP antibody (sc-2005; Santa Cruz Biotechnology, Dallas, TX) was used for detection. Imaging was performed on an LAS-4000 system (GE Healthcare Life Sciences).

MNNG/HOS and U2OS cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology) on ice for 30 min. The lysate was centrifuged at 15,000 × g, 4°C for 15 min, and the supernatant was heated at 100°C for 5 min. The concentration of the protein samples was determined using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Following separation by 10% SDS-PAGE (20 µg protein/lane), the proteins were transferred onto polyvinylidene fluoride membranes (MilliporeSigma). Subsequently, the membranes were blocked at room temperature with 5% skim milk for 1 h and incubated with primary antibodies against IARS2 (1:2,000; MilliporeSigma; SAB4502342), cleaved-caspase-3 (1:500; Cell Signaling Technology, Inc; cat. no. # 9664S), BAX (1:500; Abcam; ab32503) or β-actin (1:5,000; Abcam; ab8226) at 4°C overnight. Subsequently, the membranes were washed with Tris-buffered saline-0.5% Tween and incubated at room temperature with goat anti-mouse IgG-HRP secondary antibody (1:2,000; Santa Cruz Biotechnology, Inc; cat. no. #7076) for 2 h. Protein bands on the membranes were then detected using an ECL-PLUS/Kit (Thermo Fisher Scientific, Inc.).

Western blotting was performed to measure Bcl-2 and Bax proteins levels (23 (link)). Jurkat and KE-37 cells were incubated with different concentrations of galectin-9 (1-100 nM) for 48 h. After twice washing with ice-cold phosphate-buffered saline (PBS, pH 7.4), the cells were lysed in lysis buffer containing aprotinin (1 mg/mL), pepstatin (1 mg/mL), phenylmethylsulfonyl fluoride (PMSF) (0.05 mmol/mL), sodium orthovanadate (1 mg/mL), NaF (500 mmol/mL), and ethylenediaminetetraacetic acid (EDTA) (500 mmol/mL). The protein samples with the same volumes were loaded to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. By embedding the membranes in 5% skim milk for 2 h at room temperature, the membranes were blocked, subsequently incubated overnight at 4 °C with primary anti-Bcl-2, anti-Bax, and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, as a control, housekeeping gene; Santa Cruz Biotechnology, USA), and then were rinsed three times by PBS-Tween® 20 (PBST). Next, the membranes were shaken for 1 h at room temperature with the goat anti-mouse IgG-HRP secondary antibody (Santa Cruz Biotechnology, USA). After rinsing with PBST, the enhanced chemiluminescence detection substance (Bio-rad, USA) was used to identify target proteins.