Mouse anti gapdh monoclonal antibody

The pcDNA3.1 expression plasmid was bought from ThermoFisher (USA) and stored in our laboratory. Rabbit ARV p17 polyclonal antibody was obtained and stored in our lab. Restriction endonucleases Bam HI and Eco RI were purchased from NEB (USA). Trans1-T1 receptor cells, Taq high-fidelity DNA polymerase, pEASY-T3 cloning vector, DNA T4 ligase, and high-glucose DMEM were obtained from TransGen Biotech (Beijing, China). DL Marker and DNA gel recovery kits were purchased from TaKaRa (Dalian, China). The Animal Tissue RNA Extraction Kit and Plasmid Extraction Kit were purchased from Kang Wei Century (Beijing, China). The immunoprecipitation kit and AceQ Universal SYBR qPCR Master Mix were purchased from Novozymes Biotechnology Co. The mouse anti-Myc monoclonal antibody, rabbit anti-Flag monoclonal antibody and mouse anti-GAPDH monoclonal antibody were purchased from Beyotime Biotechnology (Beijing, China); Caspase-1 antibodies were purchased from Cell signaling technology; HRP-labeled sheep anti-mouse IgG and HRP-labeled sheep anti-rabbit IgG were purchased from Sigma; and goat anti-mouse IgG (H + L), FITC conjugate, and goat anti-rabbit IgG (H + L), PE conjugate, were purchased from TransGen (Beijing, China). FBS was purchased from LONSERA. 40,6-Diamino-2-phenylindole (DAPI) was purchased from Beyotime Company (Nanjing, China).

Protein from the sixteen samples (four groups with n = 4 per group) used in RT-qPCR was extracted from spleens with Cell Lysis Buffer for Western & IP (Beyotime, Shanghai, China) and used for expression analysis by Western blotting. Protein concentration was measured with a BCA kit (Beyotime). A 75 μg aliquot of each protein sample was loaded and then separated in SDS-PAGE 10% gels (Fdbio science, Hangzhou, China), and transferred to polyvinylidene fluoride membranes (Solarbio, Beijing, China). After blocking for 2 h at room temperature in Western Blocking Buffer (Beyotime), the membranes were incubated overnight at 4 °C with rabbit polyclonal antibody against CXCL13 (1:100 dilution; GenScript, Nanjing, China) or mouse anti-GAPDH monoclonal antibody (1:1000 dilution; Beyotime). The membranes were washed with PBS containing 0.05% Tween-20 (PBST) and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG (1:1000 dilution; Beyotime) at room temperature for 2 h. The resulting signals were visualized using FDbio-Dura ECL solution A and FDbio-Femto ECL solution B (Fdbio Science), collected by Fusion FX system (Vilber Lourmat, Marne-la-Vallée, France), and measured with ImageJ software [24 ].

The human IL‐17A recombinant protein (rhIL‐17A, #8928), rabbit anti‐ZO‐1 polyclonal antibody (#8193), rabbit anti‐MMP‐9 polyclonal antibody (#15561), MMP‐2 antibody (#4022) the rabbit anti‐Slug polyclonal antibody (#9585), the rabbit anti‐AKT polyclonal antibody (#9272), the rabbit anti‐phospho‐AKT(Ser473) polyclonal antibody (#9271), the rabbit anti‐phospho‐PI3K polyclonal antibody (#4249), the rabbit anti‐phospho‐PI3K p85 (Tyr458)/p55 (Tyr199) polyclonal antibody (#4228), the anti‐rabbit IgG HRP‐linked antibody (#7074) and anti‐mouse IgG HRP‐linked antibody (#7076) were purchased from Cell Signaling Technology (Danvers, MA, USA). The mouse anti‐α‐SMA monoclonal antibody (ab7817), the mouse anti‐Twist monoclonal antibody (ab175430), the mouse anti‐Bmi1 monoclonal antibody (ab14389) were obtained from Abcam (Cambridge, UK). The mouse anti‐GAPDH monoclonal antibody was purchased from Beyotime Biotechnology (Shanghai, China). The Alexa Fluor^® 488 fluorescent second antibody and CellTracker CM‐Dil (C7000) were purchased from ThermoFisher Scientific (Waltham, MA, USA).