Rabbit anti gapdh antibody

Rabbit anti-MS4A7 antibody (Abmart, China, #PH5636S), Rabbit anti-AKT antibody (Proteintech, China, #10,176–2-AP), Rabbit anti-GSK3β antibody (Proteintech, China, #22,104–1-AP), Rabbit anti-Phospho-GSK3β(Ser389) antibody (Proteintech, China, #14,850–1-AP), Rabbit anti-Phospho-AKT (Ser473) antibody (CST, China, #4060), Rabbit anti-GAPDH antibody (Proteintech, China, # 60,004–1-Ig).

The protein extracts from liver tissues and CFSC cells were prepared in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitor cocktails (Roche, Shanghai, China). Then, the protein samples were separated using SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Shanghai, China). After blocking with 5% skim milk, the membranes were incubated with mouse anti-α-SMA antibody (Sigma Aldrich, Shanghai, China) or rabbit anti-GAPDH antibody (Proteintech, Wuhan, China) overnight at 4 °C and subsequently incubated with HRP-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rabbit IgG (Proteintech, Wuhan, China) for 2 h at room temperature. Finally, the bands were examined with an ECL assay kit (NCM Biotech, Suzhou, China).

Human cardiac myocytes were transfected with 1 μg of WT and variant plasmid DNA. Then, we harvested the cells 48 h after transfection. We used western and immunoprecipitation lysis buffer (Beyotime, Shanghai China) with protease and phosphatase inhibitor cocktail (Beyotime) to lysis cells. The proteins were subjected to 10% SDS/PAGE and were then transferred onto nitrocellulose membranes (Millipore, Burlington, MA, USA) and immunostained with rabbit anti‐NRDG4 antibody (dilution 1 : 500; Novus Biologicals, Centennial, CO, USA), rabbit anti‐GAPDH antibody (dilution 1 : 5000; Proteintech, Chicago, IL, USA) rabbit anti‐P27 antibody (dilution 1 : 1000; Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight. The membranes were incubated with HRP‐conjugated AffiniPure Goat Anti‐Rabbit IgG(H + L) secondary antibody (dilution 1 : 10 000; Proteintech) [21, 22]. All the raw immunoblots are available in the supplement (Figure S1).

Human brain tissues from patients with TLE or traumatic brain injury, as well as rat cortices and hippocampi, were homogenized in RIPA lysis buffer (Proteintech, China). The supernatants were removed after centrifugation at 4°C (12,000 g, 15 min). Prepared proteins (40 μg per lane) were separated by SDS-polyacrylamide gel electrophoresis (SDS–PAGE; 5% spacer gel, 80 V, 30 min; 10% separating gel, 120 V, 90 min) before being transferred to a polyvinylidene fluoride (PVDF) membrane (220 mA, between 60 and 120 min according to the molecular weight of the protein). Next, the PVDF membrane was incubated at 37°C for 1 h in 10% skim milk in Tween-20 for blocking. The PVDF membrane was then incubated with polyclonal mouse anti-SAD-B-1 (diluted 1:100, Cat. No. ab206298; Abcam) diluted in 10% skim milk diluted in freshly prepared 5% BSA at 4°C overnight. The membrane was washed with Tween-20-Tris-buffered saline (TBS) for 45 min, with the solution changed every 10 min, and then incubated with goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody (1:1000, Proteintech, China) for 1 h at 37°C. A rabbit anti-GAPDH antibody (1:1000, Proteintech) was used as the loading control. Densitometry quantitation was determined using Quantity One 1-D Analysis Software (Bio-Rad Laboratories, USA) as optical density values, and the SAD-B levels were normalized to those of GAPDH.

Recombinant Human IL-7 (rhIL-7, Cat#: P5147-10 μg) and recombinant murine IL-7 (rMuIL-7 Cat#: P5930-10 μg) were bought from Beyotime. Mouse Angptl4 neutralizing monoclonal Antibodies were bought from Lexicon Pharmaceuticals.Human Angptl4-neutralizing monoclonal antibody was bought from Regeneron. Rabbit anti-IL-7 antibody (Cat:1650, for humans and mice) was bought from ABclonal. Rabbit anti-IL-7R antibody (Cat: 7626-1-AP, for human and mouse), rabbit anti-CD31 antibody (Cat: 28083-1-AP, for human and mouse), rabbit anti-ANGPTL4 antibody (Cat: 18374-1-AP, for human and mouse), rabbit anti-GAPDH antibody (Cat: 10494-1-AP, for human and mouse), rabbit IgG control antibody (Cat: 30000-0-AP), goat anti-rabbit IgG(H + L) HRP-conjugated Affinipure (Cat: SA00001-2), coralite488—conjugates affinipure goat anti-rabbit IgG(H + L), coralite594–conjugates affinipure goat anti-mouse IgG(H + L) were from proteintech.