Anti pankcr

Protein samples from PBMCs were homogenized in lysis buffer (50 mM Tris HCl, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton X-100, 0.3% NP-40, 0.1 mM PMSF and 1 µg/mL pepstatin A). Equal amounts of proteins (20 μg) were separated on 10% SDS–PAGE (Invitrogen, USA) gels, transferred onto PVDF membranes (Millipore, USA), and then blocked with 5% skim milk at room temperature for 2 h. The proteins were incubated with the following antibodies for overnight, washed with PBS, then were incubated with horseradish peroxidase-labeled secondary anti-mouse or anti-rabbit antibodies (1:2000) (ZSGB-Bio, Beijing, China) for 1 h at room temperature. Target signals were detected using the ECL detection kit (Solarbio, China). The intensities of blots were quantified by ImageJ software and relative protein levels were normalized by H3. Commercial antibodies used were as follows: anti-PanKcr (1:1000, #501), H2BK11cr (1:2000, #508), H2BK12cr (1:2000, #509), H2BK20cr (1:2000, #512) and H2BK34cr (1:2000, #514) from PTMbio, China, and anti-H3 (1:2000) from CST, USA.

For paraffin sections, dewaxing, hydration, and antigen retrieval steps were performed by following standard protocol. After blocking (10% normal goat serum [NGS]), slides were incubated with the primary antibodies as following: anti-pan-Kcr (PTM Biolabs 501 and 502, 1:1000 dilution); anti-Ehhadh (Santa Cruze sc-393123, 1:200 dilution); anti-CK7(Abcam ab181598, 1:2000 dilution); anti-Sox9 (Santa Cruze sc-166505, 1:100 dilution) overnight at 4 °C. The slides were then incubated with the secondary antibody (Goat anti-rabbit or -mouse IgG, Alexa Fluor 488 or 594; Thermo Fisher Scientific, 1:1000 dilution) for 2 h at 25 °C, followed by staining with DAPI (Thermo Fisher Scientific)/PBS for 6 min. Confocal imaging was performed using an SP8 system (Leica), and images were processed using the Leica AF software suite. For immunohistochemistry, anti-CD3 (99940, 1:150 dilution), anti-CD20 (70168, 1:500 dilution) and anti-CD68 (97778, 1:200 dilution) antibodies are from CST and anti-CD138 (Abcam ab128936, 1:500 dilution). Sections were incubated with biotinylated secondary antibodies and horseradish peroxidase avidin D (HRP, Vector). After three 5-min washes of PBS, the sections were developed with DAB kit (Vector) as standard procedures.