Af1837

Immunofluorescence was performed as previously described [2 (link),3 (link),36 (link)] with antibodies recognizing the following antigens: Myosin Heavy Chain (DSHB; MF20), Sox9 (Novus; NBP1-85551), EGFP (Aves Lab; GFP-1020), and Islet 1 (R&D; AF1837). Secondary antibodies (Jackson Immunoresearch) included anti-chicken FITC (703-095-155), anti-mouse TRITC (715-025-150), anti-goat TRITC (705-025-147), and anti-rabbit Cy5 (711-605-152). Nuclei were visualized using DAPI (Invitrogen; Slowfade Gold Antifade Reagent with DAPI; catalogue#: S36938) and fluorescence visualized using a Zeiss AxioImager II microscope.

Briefly, total proteins were extracted in RIPA (radio immunoprecipitation assay) buffer (9806; Cell Signaling, Danvers, MA, USA) containing 1 mM phenylmethylsulfonyl fluoride (8553S; Cell Signaling, Danvers, MA, USA) according to the manufacturer’s protocol. The BCA Protein Assay Kit (HX18651; Hoaxing, China) was used to measure protein concentration. Electrophoresis was performed with 30 μg total proteins separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (IPVH00010, Millipore, USA). The membrane was blocked with 5% (w/v) nonfat dry milk in 0.05 M Tris-buffered saline and 0.1% Tween-20 (TBST, pH 7.4) for 1 h and incubated with anti-ISL1(1:300; AF1837; R&D, Minneapolis, MN, USA) antibody and internal control GAPDH antibody (1:10,000; Ambion, USA) overnight at 4 °C. The secondary antibody, horseradish peroxidase-conjugated donkey anti-goat IgG (1:10,000; ab205723; Abcam, Cambridge, MA, USA), was diluted 1:5000 in TBST. The membranes were visualized using the SuperSignal West Pico Kit (Thermo Scientific, Waltham, MA, USA) substrate at room temperature. We used the ImageJ Software to assay the relative intensity of each blot. The intensity values of each group were normalized to GAPDH (internal control) in the same group.