Rabbit anti collagen 4

After treatments, cellular proteins were extracted with ice-cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCL, pH 7.5, 150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 25 mM NaF, 0.1 mM sodium orthovanadate, 10 mM NaP₄O₇, 1 nM phenylmethyl sulfonyl fluoride) containing protease inhibitors (Complete Protease Inhibitor; Roche, Manheim, Germany) on ice. Total protein concentrations were quantified by a bicinchoninic acid assay (Thermo Fischer Scientific, Waltham, MA, USA). A total of 10 μg of proteins from each sample were separated by SDS polyacrylamide gel electrophoresis on a 4% to 12% gel in MOPS running buffer (Life Technologies, Carlsbad, CA, USA), transferred onto a polyvinylidene fluoride (PVDF) membrane and probed with the following primary antibodies: rabbit anti-MMP14, mouse anti-fibronectin, rabbit anti-collagen IV, and mouse anti-GAPDH (Abcam, Boston, MA, USA). HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Invitrogen, Carlsbad, CA, USA) were used. Bound antibody complexes were detected using FluorChem E (ProteinSimple, San Jose, CA, USA). Protein expression was analyzed by densitometry using ImageJ (National Institutes of Health [NIH], Bethesda, MD, USA), and normalized to the housekeeping protein, GAPDH. All experiments were performed in triplicate for each of four donor cells.

For flatmount preparation, eyes were fixed in 4% paraformaldehyde and stained with commercial glygoprotein (Isolectin Gs-Ib4, #I21412; Life Technologies, Darmstadt, Germany) overnight at 4°C. Cryosections were fixed for 10 minutes in ethanol. The following primary antibodies were used: goat anti-CNTF-Rα (#sc-1914; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); rabbit anti-collagen IV (#ab19808; Abcam, Cambridge, MA, USA); and pStat3 rabbit mAb (#9145; Cell Signaling Technology, Inc., Danvers, MA, USA). For quantitative (q)PCR analysis, RNA was isolated from single retinas or cells grown in 6-well plates. We reverse transcribed RNA into cDNA for qPCR with the primers listed in the Supplementary Methods section. For Western blot analysis, retinal lysates were blotted onto nitrocellulose membranes and incubated with the following antibodies: anti-bActin Ab, mouse monoclonal (#A1978; Sigma-Aldrich Corp., St. Louis, MO, USA); Stat3 (79D7), rabbit mAb (#4904; Cell Signaling Technology, Inc.); pStat3 (Tyr705), and rabbit mAb (Cell Signaling Technology, Inc.). Retinal CNTF levels were determined using an ELISA set (Rat CNTF DuoSet ELISA, #DY557; R&D Systems).

The following commercial primary antibodies were obtained: rabbit monoclonal anti-TMPRSS2, rabbit anti-heat shock protein 70 (Hsp70), rabbit anti-a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), rabbit anti-EEA1, rabbit anti-collagen IV, rabbit anti-matrix metallopeptidase 16 (MMP16) and rabbit anti-tenascin C antibodies from Abcam (Cambridge, MA, USA); rabbit anti-α2-adrenergic receptor and mouse anti-Hsp90 antibodies from Sigma-Aldrich (St. Louis, MO, USA); mouse anti-signal transducer and activator of transcription 3 (STAT3), mouse anti-GAPDH, mouse anti-histone H3, rabbit anti-Rab35, rabbit anti-fibronectin, rabbit anti-phospho-STAT3^Tyr705, rabbit anti-Rab7, rabbit anti-Rab4 and rabbit anti-Rab11 antibodies from Cell Signaling (Danvers, MA, USA); mouse anti-Rab11 antibody from BD Biosciences (San Jose, CA, USA).