Anti foxp1

Infected cells were harvested and lysed in lysis buffer (#9803, Cell Signaling Technology, Danvers, MA, USA) supplemented with protease inhibitor. After centrifugation at 14,000 rpm for 10 min (4 °C), supernatants were collected, and protein concentrations were measured using the bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s protocol. Then, 20 µg of protein was separated on a polyacrylamide gel and transferred to a nitrocellulose membrane followed by incubation overnight at 4 °C with primary antibodies, anti-Ago2 (2E12-1C9, Abnova, Taipei, Taiwan), anti-MNT (#A303-626A, Bethyl Lab, Montgomery, TX, USA), anti-IRAK4 (ab32511, Cambridge, UK), and anti-FOXP1 (#2005, Cell Signaling Technology, Danvers, MA, USA)). All antibodies were diluted 1000× in 5% milk in Tris-buffered saline with Tween-20 (TBST). After incubation with the secondary and tertiary (for MNT, IRAK4, and FOXP1) antibodies and with the enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific Inc.), chemiluminescence was detected. Ago2 was quantified with Image Lab 4.0.1 software (BioRad, Hercules, CA, USA), and MNT, IRAK4, and FOXP1 were quantified using ImageJ (NIH, Bethesda, MD, USA). MNT, IRAK4, and FOXP1 protein levels were normalized relative to the total protein amount in the complete lane.

Immunoblotting and immunoprecipitation were performed according to standard protocol with the following antibodies: anti-TET2 (#36449, Cell Signaling Technology; #61389, Active Motif, 1:1000), anti-β-Actin (#A5316, Sigma, 1:5000), anti-β-Casein (#sc166530, Santa Cruz, 1:1000), anti-ERα (#ab32063, Abcam, 1:1000), anti-GATA3 (#PA520892, Thermo Fisher Scientific, 1:1000), anti-FOXA1 (sc101058, Santa Cruz, 1:1000), and anti-FOXP1 (#4402T, Cell Signaling Technologies, 1:1000). HRP-conjugated secondary antibody (#610-103-121 and #610-103-122, Rockland Immunochemicals, 1:5000).

Cellular proteins were separated by SDS-polyacrylamide gel electrophoresis (4% stacking and 10% separating gels) and then transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were blocked with 5% skim milk for 1 h and then incubated with primary antibodies (anti-FOXP1 (#4402, Cell Signaling Technology, USA), anti-Cyclin D1(#55506, Cell Signaling Technology, USA), anti-Cyclin D2(#3741, Cell Signaling Technology, USA) anti- CDK6 (#13331, Cell Signaling Technology, USA) and anti-GAPDH(#5174, Cell Signaling Technology, USA)) overnight at 4 °C. After the membranes were incubated with secondary antibodies, they were subjected to immunoblot analysis using an ECL immunoblotting kit according to the manufacturer’s protocol.