Rabbit anti mouse igg

RNA immunoprecipitation (RIP) assay was performed with Magna RIP Kit (Millipore, USA) following the manufacturer's instructions. In brief, magnetic beads were mixed with 5 μg anti‐m6A antibody (Abcam, USA), anti‐Mettl3 antibody (Abcam, USA), anti‐flag antibody (Abcam, USA), or anti‐mouse/rabbit IgG (Abcam, USA) before the addition of cell lysates (approximately 2 × 10⁷ cells for each sample). After the treatment of proteinase K, interested RNAs were eluted from immunoprecipitated complex and purified for further analysis using qPCR. Immunoprecipitated RNA was analyzed through qRT‐PCR.

The protein expressions of the NF-κB and phospho-NF-κB in kidney homogenate were obtained using the Western blot method. Thirty micrograms of kidney homogenate was separated electrophoretically in 10% sodium dodecyl sulfate polyacrylamide gel and subsequently transferred onto polyvinylidene difluoride membranes. The blots were then blocked with 5% bovine serum albumin for an hour at room temperature (RT) and probed overnight at 4 °C with 1:1000 dilution of mouse monoclonal NF-κB and phospho-NF-κB antibodies from Santa Cruz Biotechnology (Dallas, TX, USA). Next, a 1:10,000 dilution of rabbit anti-mouse IgG from Abcam (Boston, MA, USA) was used to incubate the blots at RT for 2 h. The development of the blots was done using the SuperSignal™ West Pico PLUS chemiluminescent substrate from Thermo Scientific (Rockford, IL, USA) and protein band density was measured using the image processing program ImageJ (Bethesda, MD, USA). The blots were then incubated with a 1:10,000 dilution of mouse monoclonal GAPDH or β-actin antibodies from Abcam (Boston, MA, USA), and the band density of these proteins was used as the endogenous controls.

Proteins were split from the splitting buffer and transferred to polyvinylidene fluoride or polyvinylidene difluoride membranes by electrophoresis (Millipore, Billerica, MA). The membrane was blocked with bovine serum albumin (Sigma-Aldrich, St. Louis, MO). Then, the membrane was incubated with primary antibodies at 4°C overnight, followed by incubation with secondary antibodies at room temperature for 60 min. The main antibodies include cleaved uMtCK (Abcam, dilution ratio is 1:1,000) and anti-GAPDH (Abcam, dilution ratio is 1:2,000). Secondary antibodies were rabbit anti-mouse IgG (Abcam, dilution ratio is 1:1,000). Protein bands were visualized by the ECL Chemiluminescence Detection System (Thermo Fisher Science, Rochester, NY). The experiments were repeated three times.

Immunofluorescence was carried out using standard protocol on kidney sections. Briefly, heat mediated antigen retrieval with Tris/EDTA buffer pH 6.0 was performed before commencing with staining protocol. After 30 min in BSA (G5001, Servicebio), slides were incubated overnight with specific primary antibody, rabbit anti-mouse IgG (Abcam, USA) and rat anti-mouse C3 (Santa Cruz, USA) at 4°C overnight, followed by FITC-conjugated secondary antibody (Servicebio, China). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). The samples were photographed using NIKON ECLIPSE C1.

Total proteins were collected from tissues and cultured cells using RIPA buffer (Solarbio, Beijing, China). The concentrations of proteins were measured using bicinchoninic acid (BCA) assay kit (Beyotime, Biotechnology, Shanghai, China). The proteins were separated using 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and electro-transferred on to polyvinylidene fluoride (PVDF) membrane (Reno, Hangzhou, China) in a semi-dry blotting apparatus (Bio-Rad, Hercules, California). After blocking with TBST-soluble 5% dried skimmed milk at room temperature for 2 h, the membranes were respectively bound to the mouse monoclonal anti-human SOX12 antibody (1:1000; Abcam, Cambridge, MA, U.S.A.) or mouse monoclonal anti-human GAPDH (1:5000; Santa Cruz Biotechnology Inc, CA, U.S.A.) at 4°C overnight. Then membrane was bound to the corresponding secondary antibodies (Rabbit anti-mouse IgG, 1:6000; Abcam) at 37°C for 1 h. The blots were assessed using an enhanced chemiluminescence reagent (ECL, GE Healthcare, Chicaogo, IL, U.S.A.).

IHC was performed with antibodies directed against B220/CD45R (RA3-6B2; R&D Systems; 1:40 dilution), CD138 (281-2; BD Biosciences; 1:50 dilution), CD3 (A0452; DAKO; 1:100 dilution), myeloperoxidase (A0398; DAKO; 1:100 dilution), Ki-67 (RM-9106-S1; Thermo Fisher Scientific; 1:200), Bcl6 (sc-858; Santa Cruz Biotechnology; 1:50), Irf4 (sc-6059; Santa Cruz Biotechnology; 1:100), and Pten (M362729-2; Agilent; 1:150 dilution). Pre-treatment of sections was conducted with EDTA for 20 min (B220, CD138, CD3, myeloperoxidase), 30 min (Ki-67, Irf4, Pten), or 40 min (Bcl6).
We used goat anti-rabbit, rabbit anti-rat, rabbit anti-goat, and rabbit anti-mouse secondary antibodies (AffiniPure Goat Anti-Rabbit IgG (H+L) [111-005-003; Jackson ImmunoResearch], AffiniPure Rabbit Anti-Rat IgG [312-005-045; Jackson ImmunoResearch], Rabbit Anti-Goat IgG [P0449; DAKO], and Rabbit Anti-Mouse IgG [ab125904; Abcam]).