Anti itgb2

BMDMs were lysed in 1×SDS sample buffer. Kidneys were lysed with RIPA buffer containing 1% NP-40, 0.1% SDS, 100 mg/ml PMSF, 1% protease inhibitor cocktail, and 1% phosphatase I and II inhibitor cocktail (Sigma-Aldrich) on ice. The supernatants were collected after centrifugation at 16,000 g at 4 °C for 30 min. Protein concentration was determined by bicinchoninic acid protein assay (BCA Protein Assay Kit, Pierce Thermo-Scientific, Rockford, IL) according to the manufacturer’s instruction. The primary antibodies were anti-PP2Ac (cat: 2038, Cell Signaling Technology, Boston, MA, USA, 1:1000), anti-PP2Acα (cat: ab106262, Abcam, Cambridge, UK, 1:1000), anti-PP2Acβ (cat: ab168371, Abcam, 1:1000), anti-methyl-PP2Ac (L309) (cat: ab66597, Abcam, 1:1000), anti-Itgb2 (cat: ab119830, Abcam, 1:1000), anti-Epac1 (cat: ab124162, Abcam, 1:1000), anti-FN (cat: F3648, Sigma-Aldrich, 1:10000), anti-Stat6 (cat: ab32520, Abcam, 1:1000), anti-p-Stat6 (T645) (cat: BS4186, Bioworld Technology, Nanjing, China, 1:1000), anti-Rap1a/b (cat: 4938, Cell Signaling Technology, 1:1000), anti-tubulin (cat: sc53646, Santa Cruz Biotechnology, 1:10000), and anti-GAPDH (cat: FL-335, Santa Cruz Biotechnology, 1:5000). Quantification was performed by measuring the intensity of the signals with the aid of the National Institutes of Health ImageJ software package.

Kidney cryosections at 3-μm thickness were fixed with 4% paraformaldehyde for 15 min followed by permeabilization with 0.2% Triton X-100 in 1×PBS for 5 min at room temperature. After blocking with 2% donkey serum for 60 min, the slides were immune-stained with the following antibodies: anti-FN (cat: F3648, Sigma-Aldrich), anti-F4/80 (cat: 14–4801, eBioscience, San Diego, CA, USA), anti-CD3 (cat: 555273, BD Pharmingen, New Jersey, USA), anti-PP2A C subunit (cat: 2038, Cell Signaling Technology), anti-Stat6 (phospho-T645) (cat: BS4186, Bioworld Technology), anti-TNFα (cat: BS1857, Bioworld Technology), anti-Laminin (cat: ab44941, Abcam, 1:100), anti-Itgb2 (cat: ab119830, Abcam, 1:200), and anti-CD68 (cat:MO876, Dako). Tissues were stained with DAPI to visualize the nuclei. Slides were viewed with an OLYMPUS DP74 and BX53 Epifluorescence microscope equipped with a digital camera. The number of F4/80-positive and CD3-positive macrophages was counted from ten randomly selected fields in the cortical area for each sample under microscope (400×), and an average number of positive cells for each section was calculated.