Anti rabbit hrp secondary

Passage 7 cardiac PCs were grown in a 96‐well plate (PerkinElmer Cell Carrier, Perkin Elmer, Waltham, MA, USA) until 90% confluent. Cells were washed with warm 1× DPBS and fixed with 4% paraformaldehyde (Sigma) for 30 min at room temperature. Cells were washed three times with 1× DPBS and permeablized with 0.1% Triton X‐100 (Sigma) for 10 min at room temperature. Cells were then blocked with SuperBlock (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. After blocking, anti‐hypoxia‐inducible factor 1 alpha (HIF‐1α) antibody (Abcam, Cambridge, UK) diluted at 1 : 100 with SuperBlock was added and incubated at 4 °C overnight. Next day, cells were washed three times with wash buffer (0.1× SuperBlock + 1× DPBS). Secondary anti‐rabbit HRP (Cell Signaling Technologies, Danvers, MA, USA) diluted 1 : 1000 in SuperBlock was added and incubated for 2 h at room temperature in the dark. Secondary antibody was washed off three times with wash buffer. A 1 : 1000 dilution of 300 µm DAPI (Life Technologies, Carlsbad, CA, USA) stain was added on for 5 min at room temperature. Cells were washed three times with 1xDPBS and mounted with ProLong Diamond (Life Technologies) mounting media. Cells were imaged on an Opera Phenix confocal (PerkinElmer) at 40×.

The following antibodies were used in this study: VINCULIN (Cell Signaling 13901), ACTIN (Santa Cruz sc-47778), GAPDH (Cell Signaling 5174), GFAT1 (Abcam 125069), NAGK (Atlas Antibodies HPA035207), O-GlcNAc (Abcam 2735), panCK (Cytokeratin, DAKO M3515), biotinylated HABP (Calbiochem 385911), secondary anti-mouse-HRP (Cell Signaling 7076), and secondary anti-rabbit-HRP (Cell Signaling 7074).

Protein concentrations were determined using the BCA Protein Assay Kit (23225, Thermo Fisher Scientific). The following antibodies were used: BRCC3: ab108411, Abcam, Cambridge, UK; Tubulin: T5168, Sigma-Aldrich; ABRO1: ab74333, Abcam; Abraxas: ab139191, Abcam; BABAM1: sc-160990, Santa Cruz, Dallas, TX, USA; UIMC1: 14466S, New England Biolabs, Ipswich, MA, USA; FLAG-tag (DYKDDDDK): 2368, Cell Signaling Technology, Danvers, MA, USA; HA-tag (YPYDVPDYA): 901533, BioLegend; G-CSF: sc-53292, Santa Cruz; β-Actin HRP: ab20272, Abcam; secondary anti-mouse HRP: 7076S, Cell Signaling; secondary anti-rabbit HRP: 7074S, Cell Signaling.