Alkaline phosphatase conjugated anti rabbit igg

The mitochondrial protein concentration was measured by BCA Protein Assay Reagent Kit (Beyotime, Nantong, China). Samples containing total mitochondrial protein were separated by 10% (w/v) SDS-PAGE and transferred onto a PVDF membrane (Millipore, Bedford, MA, United States) (details are in Western Blotting Assay section). The following antibodies were used: Antibodies for PDK4 (Cat# 12949-1-AP), HADHA (Cat# 10758-1-AP), Annexin 6 (Cat# 12542-1-AP), NNT (Cat# 13442-2-AP), and COX-IV (Cat# 66110-1-Ig) were obtained from Proteintech (Wuhan, Hubei, China). The secondary antibody (alkaline phosphatase-conjugated anti-rabbit IgG) was from Promega Corporation (Madison, WI, United States). The intensities of the protein bands were quantified using a Bio-Rad ChemiDocTM EQ densitometer and Bio-Rad Quantity One software (Bio-Rad, Hercules, United States).

Separated proteins were blotted on polyvinylidene difluoride membranes by semi-dry transfer. Target proteins were detected with the antibodies as follows: anti-Fra a 1e with 6× His (1:400 dilution) and alkaline phosphatase-conjugated anti-guinea pig IgG (1:3000 dilution; ab102367, Abcam, Cambridge, UK) were used to identify Fra a 1. The anti-Fra a 2 synthetic peptide (1:500 dilution) and alkaline phosphatase-conjugated anti-rabbit IgG (1:2000 dilution; S373B, Promega) were used to specifically identify Fra a 2. The BCIP-NBT Solution Kit (Nacalai Tesque) was used for alkaline phosphatase staining.

Rabbit anti-PgDPP7 antiserum was prepared as previously reported [21] (link). Recombinant DPPs or cell lysate whole proteins were separated on SDS-PAGE, then subjected to Coomassie brilliant blue (CBB) staining. Alternatively, separated proteins were transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany), and subjected to immunoblotting with anti-PgDPP5 [16] (link) or anti-PgDPP7 serum. Protein bands were incubated with alkaline phosphatase-conjugated anti-rabbit IgG (Promega, Madison, WI), and visualized with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. To detect PeDPP7, blotted membranes were incubated with anti-PgDPP7 serum, then with a peroxidase-conjugated anti-rabbit IgG antibody (Promega), and the color was finally developed with 4-chloro-1-naphthol to avoid development of a nonspecific 100-kDa band.