β actin or gapdh antibody

NRCM were lyzed in 100 µl RIPA buffer (containing 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM phenylmethylsulphonyl fluoride, 500 ng/ml Leupeptin, 1 mg/ml Aprotinin and 2.5 mg/ml Pepstatin A). Western blot analysis was conducted using standard protocols. Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were blocked in 3% BSA and incubated with primary antibodies against IL-10R1 (Santa Cruz), phosphorylated STAT3 or total STAT3 (Cell Signaling) overnight, followed by HRP-linked anti-rabbit secondary antibody (Cell Signaling) for 2 h. Proteins were visualized using enhanced chemiluminescence (GE Healthcare) on a ChemiDoc XRS Imaging System (Biorad). Membranes were then incubated with β-actin or GAPDH antibody (abcam) as loading control.

For western blot analysis of erythrocyte PMCA expression, blood was collected from the jugular vein of PMCA4^−/− and WT mice under anaesthesia with 2.5% isoflurane, and heparinized. Blood was centrifuged at 1800g for 5 min and the plasma and buffy coat were discarded. Erythrocytes were then washed 4 times in twice their volume of 0.9% saline, with centrifugation for 5 min at 1200g. Packed erythrocytes were then lysed in 10 × their volume of RIPA buffer (containing 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM phenylmethylsulphonyl fluoride, 500 ng/ml Leupeptin, 1 mg/ml Aprotinin and 2.5 mg/ml Pepstatin A), and 10 µl of the resultant lysate was separated by SDS-PAGE and transferred to PVDF membranes (Millipore) using standard protocols.
Membranes were blocked in 3% BSA (for PMCA4 detection) or 3% non-fat milk (for PMCA1 detection) for 1 h at room temperature, before refrigerated incubation overnight with primary antibodies against PMCA4 (clone JA9-Abcam) or PMCA1 (clone F-10-Santa Cruz). Proteins were visualized the following day using enhanced chemiluminescence (GE Healthcare), after incubation with HRP-linked anti-mouse or anti-rabbit secondary antibody (Cell Signalling) on a ChemiDoc XRS Imaging System (Biorad). Membranes were then incubated with β-actin or GAPDH antibody (abcam) as loading control.