Anti med1

Mammary tissues were harvested on day 1 of lactation (L1), fixed in 10% formalin, dehydrated using an ethanol series and xylene, and embedded in paraffin according to standard protocols. Paraffin blocks were sectioned at 4 μm thickness. For immunohistochemistry, antigen unmasking was performed in a TintoRetriever Heat Retrieval System (Bio SB Inc., Goleta, CA, USA) using 10 mM sodium citrate buffer (pH 6.0) for 10 min. Sections were blocked by incubating with 3% normal goat serum at room temperature for 1 h. Tissues were incubated overnight at 4 °C with the following primary antibodies (diluted 1:100): anti-STAT5A (#LS-C386212-100; LSBio, Seattle, CA, USA); anti-ELF5 (#70R-49702; Fitzgerald, Kampenhout, Belgium); anti-NFIB (#39091; Active Motif, Carlsbad, CA, USA); anti-GR (#PA1-511A; Invitrogen); anti-MED1 (#A300-793A; Bethyl Laboratories, Waltham, MA, USA); and anti-E-cadherin (#610181; BD Biosciences, Franklin Lakes, NJ, USA). After washing, tissues were incubated with Alexa Fluor 488-conjugated anti-mouse IgG (#R37120; Invitrogen) or Alexa Fluor 594-conjugated anti-rabbit IgG (#R37117; Invitrogen) for 1 h at room temperature. Slides were then mounted with ProLong Gold Antifade Mountant with DAPI (Invitrogen), imaged under a Zeiss LSM 800 confocal microscope, and analyzed using the ZEN image browser.

The total proteins were extracted using RIPA lysis buffer. The following dilutions of antibodies were used for each antibody: anti-MyoG (1:2000, sc-576, Santa Cruz), anti-MyHC (1:2000, M4276, Sigma), anti-hnRNPL (1:5000, sc-28726, Santa Cruz), anti-hnRNPK (1:5000, 4675, Cell Signaling), anti-MED1 (1:5000, A300–793A, Bethyl Laboratories), anti-RAD21 (1:5000, A300–080A, Bethyl Laboratories), anti-RBBP5 (1:5000, A300–109A, Bethyl Laboratories), anti-YY1 (1:2000, sc-1703, Santa Cruz), anti-MyoD (1:2000, sc-760, Santa Cruz), anti-α-Tubulin (1:5000, sc-23948, Santa Cruz), and anti-H3K36me3 (1:5000, ab9050, Abcam). For Immunofluorescence staining of cultured C2C12 cells, the following dilutions were used: anti-MyHC (1:350, M4276, Sigma). All fluorescent images were captured with a Nikon fluorescence microscope.