Anti lhx4

Wild-type retinas at P8 were collected and lysed in the RIPA buffer (ThermoFisher, #89900) with a protease inhibitor cocktail (Roche, #04693124001). Three biological replicates were performed. Cell lysate was centrifuged for 20 min at 12,000 rpm at 4°C. The super-natant was incubated with anti-ISL1 (Abcam Cat# ab20670, RRID:AB_881306) at 4°C overnight. Then, the protein A/G agarose beads (Santa Cruz Biotechnology Cat# sc-2003, RRID:AB_10201400) was added into the sample and incubated at 4°C for 4 hours. After four washes with RIPA buffer, the sample was suspended with 2x Laemmli Sample Buffer (Bio-Rad #1610737) and boiled for 10 min. Then, the sample was loaded into SDS-PAGE gel, and ran for 2 hours at 100 V. The proteins were transferred from the gel to PVDF membrane using Mini Trans-Blot electrophoretic transfer cell (Bio-Rad). Next, the membrane was blocked with the blocking buffer (5% milk in TBST) for 1 hour at RT, and incubated with anti-LHX4 (1:1000, Proteintech # 11183–1-AP) at 4°C overnight. Then, the membrane was washed three times with TBST for 5 min each, it was incubated with VeriBlot for IP Dectection Reagent (1: 1000, Abcam #ab131366) for 1 hour at RT. After the membrane was washed four times with TBST for 5 min each, detection was done with ImageQuant LAS4000 biomolecular imager (GE Healthcare).

All tissue processing and immunofluorescence experiments were performed as in Emerson and Cepko16 (link) and Buenaventura et al.3 (link) Antibodies used are the following [antibody (host, vendor, catalogue #, dilution, RRID)]: anti-Otx2 (goat, Novus Biologicals, Centennial, CO, USA; AF1979, 1:500, AB_2157172), anti-Rxrg (mouse, Santa Cruz Biotechnology, Dallas, TX, USA; sc-365252, 1:50, AB_10850062), anti-Lhx4 (rabbit, Proteintech, Rosemont, IL, USA; 11183-1-AP, 1:250, no RRID), anti-Lhx3/4 (mouse, Developmental Studies Hybridoma Bank [DSHB], Iowa City, IA, USA; 67.4E12, 1:200, AB_2135805), anti-Nr2e3/PNR (mouse, R&D Systems, Minneapolis, MN, USA; PP-H7223–00, 1:250, AB_2155481), anti-β-galactosidase (mouse, DSHB; 40-1a-s, 1:20, AB_528100), rabbit anti-PKC-alpha (rabbit, Sigma-Aldrich, St. Louis, MO, USA; P4334, 1:500, AB_477345), anti-MAFA/L-MAF (rabbit, gift from Celio Pouponnot, 1:500, no RRID), anti-RAXL (rabbit, gift from Hajime Ogino, 1:500, no RRID), anti-Cone Arrestin (rabbit, Millipore Sigma, Burlington, MA, USA; AB15282, 1:2,000, AB_1163387), and anti-GFP (chicken, Abcam, Cambridge, MA, USA; ab13970, 1:2000, AB_300798). RRID = Repository Resource ID from The Antibody Registry (http://antibodyregistry.org, in the public domain).