Anti pp2a c subunit

Retinae were isolated and homogenized in buffer [80 mm Tris, pH 8.0, 4 mm MgCl₂, and 0.5 mg/ml protease inhibitor cocktail (Roche)] and centrifuged at 30,000 × g for 10 min. The supernatant was collected (soluble fraction), and the pellet was solubilized using Triton X-100-containing buffer [80 mm Tris, pH 8.0, 4 mm MgCl₂, 1% Triton X-100, and 0.5 mg/ml protease inhibitor cocktail (Roche)]. Protein was quantified using Bradford protein assay (Bio-Rad). Equal amount was loaded per lane and separated in 12% Bis-Tris SDS-PAGE gel (Invitrogen) and transferred onto nitrocellulose membranes. The blots were blocked in TBS-T buffer (20 mm Tris, pH 7.5, 136.8 mm NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk. The following primary antibodies were used: anti-PP2A A subunit (1:500, #2041, Cell Signaling), anti-PP2A C subunit (1:2000, #2259, Cell Signaling), and rabbit polyclonal anti-Gβ5 (1:2000) (Watson et al., 1996 (link)). The secondary antibodies IRDye 680 goat anti-mouse and IRDye 800 goat anti-rabbit antibodies (LI-COR Biosciences) were used. The proteins were visualized and quantified using Odyssey Infrared Imaging System (LI-COR Biosciences).

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.