Phosphatase inhibitors

Proteins were isolated using RIPA buffer (Boston BioProducts, BP-115) with protease inhibitor (Roche, 4693132001) and phosphatase inhibitors (New England Biolabs, P0758L). Protein concentrations were determined using Pierce BCA assay (Thermo Fisher Scientific). A total of 20 μg protein were loaded per lane on a 4%–20% Mini-PROTEAN TGX Gel (Bio-Rad, 456-1096). Separated proteins were transferred to PVDF membranes using the Transfer Turbo Blot system (Bio-Rad) and Trans-Blot Turbo RTA Transfer Kit (Bio-Rad, 170-4272). The membrane was blocked with 5% nonfat milk in TBST for 1hour at room temperature. After blocking, the membrane was incubated overnight at 4°C with antibodies against Flag Tag (Cell Signaling Technology, 2368, 1:1000), GAPDH (Cell Signaling Technology, 2118, 1:4000), VCAM-1 (Cell Signaling Technology, sc-13160, 1:1000), ICAM-1 (R&D Systems, Bio-Techne, BBA3), IκBα (Cell Signaling Technology, 4812, 1:1000), β-actin (Cell Signaling Technology, 4970, 1:3000), and phospho-IκBα (Cell Signaling Technology, 2859, 1:1000), IL-1β (Abcam ab9722, 1:1000), MCP-1 (Abcam ab25124, 1:1000), COX-2 (Cell Signaling Technology 12282p), p-P38MAPK (Cell Signaling Technology 4511L, 1:1000), and P38 MAPK (Cell Signaling Technology 9212L, 1:1000). Quantification of protein bands was performed using a luminescent image analyzer (Bio-Rad, Chemidoc).

Retinas were dissected in ice cold, sterile PBS containing protease inhibitors (#20-201, Millipore, Billerica, CA) and phosphatase inhibitors (#P0758S New England Biolabs, Ipswich, MA). To preserve protein following collection, retinas were flash frozen in liquid nitrogen and stored at −80 °C until further use. Proteins were prepared by sonicating retinas, and clarification by centrifugation at 12,000 × g. Protein concentrations were measured using the BCA assay (ThermoFisher, Waltham, MA), and western blots were done as previously described^{31 (link)}. Antibodies used: pSTAT3 Y705 (1:2,000 Rabbit, #9145), total STAT3 (1:2,000, Rabbit, #9132 L) (Cell Signaling Technology, Danvars, MA), and actin (Goat, ab6276, Abcam, Cambridge, UK). The antibodies for phosphorylated and total STAT3 have been extensively characterized^{3 (link),4 (link),58 (link)}. Signal detection was done using fluorescently-labeled secondary antibodies to rabbit (Goat, IRDye 800 CW) and mouse IgGs (Goat, IRDye 680RD) (LICOR, Lincoln, NE). Fluorescent signals were measured using a LICOR Odyssey imager and Odyssey Image Studio software (version 5.2). Western blots in Fig. 2 were cropped. Full, uncropped versions of these blots can be found in Figs S7 and S8.