Complete mini protease inhibitors and phosphatase inhibitors

Streptococcus suis strains 05ZYH33 and 05ZYH33Δmrp were pre-incubated with hFg for 1 h and added to hCMEC/D3 cell monolayers at a MOI of 100:1 for indicated time. Cell monolayers were washed three times with ice-cold PBS and lysed with 50 mM Tris pH 7.5, 10 mM MgCl₂, 0.5 M NaCl, 2% Igepal, complete mini protease inhibitors and phosphatase inhibitors (Roche) on ice for 20 min. Cell lysates were separated by SDS-PAGE electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies and appropriate HRP-conjugated secondary antibodies. The protein signals were developed with SuperSignal West Dura Extended Duration substrate (Pierce) and imaged using ChemiDoc^TM XRS+ system (Bio-Rad).

Whole-cell lysates were prepared from snap frozen cell pellets using RIPA lysis buffer with Complete Mini Protease inhibitors and Phosphatase inhibitors (Roche). Nuclear and Cytoplasmic extracts were prepared from cell pellet or tissue sample using NE-PER™ Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific). Equal amount of the whole cell lysates or nuclear/cytosolic extracts were loaded and separated by SDS–PAGE and transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA), followed by immunoblotting analysis with antibodies to EHMT2/G9a (abcam, ab185050), Di-Methyl-Histone H3 (cell signaling, #4658), Histone H3K9me1 (santa cruz, #39681), Histone H3 (cell signaling, #4499), Tri-Methyl-Histone H3 (cell signaling, #4909), β-actin (Sigma, A2228), phospho-RelB (cell signaling, #5025), RelB (cell signaling, #10544), Bim (cell signaling, #5392), NF-κB2 (cell signaling, #3017), HDAC4 (cell signaling, #5392), HRP-conjugated anti-Rabbit (Cell Signaling #7074), and HRP-conjugated anti-Mouse (Cell Signaling #7076). All primary antibodies were used at 1:2000 dilution and secondary antibody used at 1:10,000 dilution. The blots were developed using ChemiDoc™ MP Imaging System (Bio-rad).