Ab13248

Podocytes were lysed using RIPA buffer, and protein concentration was determined using the BCA protein assay kit. Approximately 30 μg of protein from each sample was separated using a 10% SDS-polyacrylamide gel and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in TBST and incubated with primary antibodies overnight at 4°C. Membranes were then incubated with the corresponding secondary antibodies for 1 h at room temperature and washed in TBST. Proteins were detected using specific antibodies: HMOX1: Abcam, ab13248; Sirt1: Abcam,ab110304; LC3B:Abcam,ab63817; AMPK:Abcam,ab110036; p-AMPK: Abcam,ab194920; P62: Abcam,ab56416; beta-actin: Abcam: ab115777.

The following antibodies were used:
Rabbit monoclonal anti-EGFR (ab52894, Abcam, Cambridge, UK); mouse monoclonal anti-EGFR (sc-120, Santa Cruz Biotechnology, USA); mouse monoclonal anti-EGFRvIII (L8A4, Absolute Antibodies, UK); rabbit polyclonal anti-EGFR (ab5652, Abcam, UK); mouse monoclonal anti-HO-1 (ab13248, Abcam, UK); mouse monoclonal anti-β-actin (AC-15, Sigma-Aldrich, USA); IRDye^® 800CW goat anti-mouse IgG (827-08364, LI-COR Biotechnology, Germany); IRDye^® 680RD goat anti-rabbit IgG (926-68071, LI-COR Biotechnology, Germany).

The ears of three 8-month-old transgene-positive goats were injected intradermally with 100 μL of 3 mg/mL Pam3CSK4, after which the tissues were collected at 1, 8, and 48 h [18 (link), 19 (link)]. Samples were fixed with 4% paraformaldehyde and embedded in paraffin. Hematoxylin and eosin staining was used to investigate inflammatory responses and immunohistochemistry was used to detect HO-1 protein expression (Abcam, ab13248, Cambridge, UK). Six fields from each slide were randomly selected. Optical densities were quantified by scanning densitometry and expressed in arbitrary units determined by Image J software (NIH, USA).

Partial kidney tissues were thoroughly homogenized in a lysis buffer (0.97% protease inhibitor cocktail, 0.94% 50 mM phenylmethylsulfonyl fluoride, and 97.09% 1x RIPA) on ice. The protein concentrations of the homogenates were measured using the BCA Protein Assay Kit (Merck Millipore, USA); 50 μg of protein was electrophoresed on 12% SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membrane (Merck Millipore, USA), and blocked in 5% bovine serum albumin (BSA) in Tris-buffered saline. Then, the bands were incubated overnight at 4°C in a corresponding primary antibody solution containing Nrf2 (ab137550), HO-1 (ab13248), SOD1 (ab13498), SOD2 (ab13533), and CAT (ab16731) (1 : 2000; Abcam, UK) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ABS16, 1 : 2000, Merck Millipore, USA) and then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (bs-0295G, 1 : 2000, Beijing Biosynthesis Biotechnology Co. Ltd., China) for 4 hours at 4°C. Specific signals were visualized with ECL detection on a gel imaging system (UVP, California, USA). The average optical density of the bands was quantified using ImageJ (National Institutes of Health, Bethesda, USA).

Western blots were performed as previously described [25 (link)]. Total protein was extracted from kidney tissue in RIPA lysis buffer (25 mM Tris-HCl, 25 mM NaCl, 0.5 mM EDTA, 1% Triton X-100, and 0.1% SDS) with 1% PMSF protease inhibitors (P1005, Beyotime Biotechnology, China) and phosphatase inhibitors (P1081, Beyotime Biotechnology, China) added. Equal amounts of protein were separated by 10-15% SDS-PAGE and then transferred to PVDF membranes (IPVH00010, Millipore, Germany). After blocking with 5% nonfat milk for 3 h, the membranes were incubated with the primary antibodies including TGF-β1, α-SMA, Nrf2, HO-1, NQO1, ASC, caspase-1 (ab215715, ab32575, ab137550, ab13248, ab80588, ab175449, ab1872, Abcam, UK), p-Smad2, Smad2, p-Smad3, Smad3, E-cadherin, NLRP3 ((#18338, #5339, #9520, #9523, #3195, #15101, Cell Signaling Technology, USA), and cleaved caspase-1 (AF4005, Affinity Biosciences, USA) at 4°C overnight. Then, the membranes were washed 3 times with TBST and incubated with the secondary antibodies for 1 h at room temperature. After washing with TBST for a further three times, the protein bands were visualized by enhanced chemiluminescence (32134, Thermo, USA) solution and imaged with Automated Imaging System (Gene Gnome5, Synoptics Ltd, UK). GAPDH or β-actin was assumed to be similarly abundant in all samples and was used as a loading control.

Cells were fixed with 4% formaldehyde and pre-incubated with 10% normal goat serum (710,027, KPL, USA), and then incubated with primary antibodies anti-Bach2 (ab83364, Abcam) and anti-HO-1 (ab13248, Abcam). Secondary antibodies were fluoresce-labeled antibody to rabbit IgG (021516, KPL, USA) and rhodamine-labeled antibody to mouse IgG (031806, KPL, USA). DAPI (157,574, MB biomedical) was used for nuclear counter staining. Images were captured by confocal microscopy (DM6000 CFS, Leica) and processed by LAS AF software.