Rabbit anti mouse β actin antibody

Isolated crypts or IESC samples were incubated in RIPA buffer (Thermo Scientific, Carlsbad, CA, USA). The protein samples were separated by 12% SDS-PAGE and transferred to PVDF membranes. The membranes were probed overnight at 4 °C with the following primary antibodies: rabbit anti-mouse Dnmt1 antibody (1:2000; Abcam, Cambridge, MA, UK), rabbit anti-mouse Dnmt3a antibody (1:1000; Abcam), rabbit anti-mouse Sox9 antibody (1:1500; Abcam), and rabbit anti-mouse β-actin antibody (1:2000; Cell Signaling Technology, Danvers, MA, USA). The data were analyzed using relative intensity with β-actin as the internal control. The Western blot experiments were performed in three repetitions using independent samples.

Cells were seeded at 2 × 10⁵ cells/ml in a four-well chamber slide and incubated overnight. After 24-h treatment with 100 ng/ml CCL2 and 10 ng/ml LPS, cells were fixed with ice-cold methanol for 10 min, washed with PBS/0.01% Triton-X, and blocked with PBS/3% BSA. Cells were incubated with rabbit anti-mouse-β-actin antibody (Cell Signaling Technology, Beverly, MA) for 1 h followed by incubation with secondary antibodies Alexa Fluor 555-conjugated anti-rabbit IgG (H+L) F(ab’)₂ (Cell Signaling Technology). Cells were washed with PBS/0.01% Triton-X again before counterstained with Prolong gold antifade DAPI reagent. Slide was viewed in a Leica TCS SP5 II confocal microscope (Leica Microsystems, Mannheim, Germany).