Mouse α flag

Chromatin immunoprecipitation (ChIP) was performed with the ChIP-IT Express Kit (Active Motif) using 10⁷ cells per ChIP. For the Flag ChIP, αTC1-6 cells and βTC6 cells were infected with Flag-Grg3 lentivirus. ChIP was performed with mouse-α-Flag (Sigma-Aldrich) antibody on Flag-Grg3–infected and GFP-infected cells. For the Nkx6.1 ChIP, Nkx6.1 antibody (BCBC) was used in both αTC1-6 and βTC6 cells. ChIP-qPCR product was quantitated using 2^{(CTinput – CTChIP)}. Results are shown as ChIP/input in relative units. PCR was performed on ChIP products for the glucagon promoter (5′-AAGCAGATGAGCAAAGTGAGTG-3′ and 5′-AGGCTGTTTAGCCTTGCAGATA-3′), Hnf1β promoter (5′-CTCTGGCAAGTCCCA ATCCC-3′ and 5′-CCATGATCTCCACCATTAGGC-3′), and Hnf6 promoter (5′-TTTGGGCCATGACATAGTTTC-3′ and 5′-CTTGCTACCTCCTGGTCTTCC-3′) using Power SYBR Green PCR Master Mix (Applied Biosystems) on a StepOnePlus Real-Time PCR System (Applied Biosystems).

For co-immunoprecipitation, cell lysates were prepared in the lysis buffer (50mM Tris-HCl, pH7.5; 150mM NaCl, 1mM EDTA, 1mM EGTA, 5mM Na₄P₂O₇, 25mM NaF, 1% Triton X-100) with protease inhibitors (cocktail) on ice for 30min. After clarification by centrifugation, the lysates were incubated for 2-6h at 4 °C with antibodies pre-bound to protein A/G agarose beads. Beads were washed four times with washing buffer (50mM Tris-HCl, pH7.5; 150mM NaCl, 1mM EDTA, 1mM EGTA, 5mM Na₄P₂O₇, 25mM NaF, 0.5% Triton X-100), and eluted in 1x SDS loading buffer. Eluted samples were analyzed by western blotting. Antibodies used for immunoprecipitation were: mouse α-Myc (MBL International), mouse α-V5 (Thermo Fisher Scientific), mouse α-TEAD1 (BD Biosciences), EZview™ Red Anti-HA Affinity Gel (Sigma-Aldrich) and EZview™ Red Anti-Flag Affinity Gel (Sigma-Aldrich). Primary antibodies used for western blot were: rabbit α-E2F1 (X. Bi, 1:2000), mouse α-Flag (Sigma-Aldrich, 1:10000), rabbit α-Yki (L. Zhang, 1:5000), mouse α-TEAD1 (BD Biosciences, 1:500), mouse α-HA (Santa Cruz Biotechnology, 1:1000), mouse α-V5 (Thermo Fisher Scientific, 1:20000), mouse α-Myc (MBL International, 1:2000), rabbit α-hE2F1 (CST, 1:1000), mouse α-GAPDH (CWBiotech, 1:1000) and mouse α-β-actin (Proteintech Group, 1:5000).