Anti histone h3

GPS cells were transfected with pTroIGFBP5b-WT-N3, pTroIGFBP5b-ΔHBM-N3, pTroIGFBP5b-ΔSP-N3, pTroIGFBP5b-Δ(HBM+SP)-N3, or pEGFPX-N3 in 10-cm-diameter culture dishes. Nuclear and Cytoplasmic Extraction Reagent Kit (Beyotime, Beijing, China) was used to separately extract nuclear and cytoplasmic proteins. After protein separation by 15% SDS-PAGE and transfer to a PVDF membrane (Millipore, Germany), the membrane was blocked with 5% BSA for 1 h. Then, the membrane was incubated with anti-EGFP (1/2,000 dilution, Bioss, Beijing, China) at 4°C overnight. After 10 min of washing with TBST three times, the secondary antibody (HRP-conjugated goat anti-mouse IgG and 1/2000 dilution) was added and incubated for 1 h at RT. Anti-β tubulin and anti-Histone H3 (Bioss, Beijing, China) were used as the nuclear and cytoplasmic internal references, respectively. The experiment was performed in triplicate.

To evaluate the TroBcl2 protein overexpression and knockdown efficiencies, total protein from GPS cells transfected with pTroBcl2 or siTroBcl2 was extracted and analyzed using western blotting with a mouse anti-His (monoclonal, 1:1,000 dilution) primary antibody and HRP-conjugated goat anti-mouse IgG (1:2,000 dilution) as the secondary antibody. An anti-β-actin mouse monoclonal antibody (Bioss, Beijing, China) was used as an internal reference. Immunoreactions were detected with a supersensitive ECL substrate (Biosharp, Anhui, China).
To detect the subcellular localization of TroBcl2, GPS cells were transfected with pTroBcl2-N3 or pEGFPX-N3. The Nuclear and Cytoplasmic Extraction Reagents Kit (Beyotime, Beijing, China) was used to separately extract the nuclear and cytoplasmic proteins. The primary antibodies were mouse anti-TroBcl2 polyclonal antibody (1:2,000 dilution), and the secondary antibody was HRP-conjugated goat anti-mouse IgG (1:2,000 dilution, Bioss, Beijing, China). Anti-Tubulin and anti-Histone H3 (Bioss, Beijing, China) were used as the nucleus and cytoplasm internal reference, respectively.

GA was purchased from Meilun Biological Co., Ltd. (Dalian, China), and dissolved in 10% DMSO at a concentration of 5 mg/mL. The chemical structure of GA is shown in Figure 1. The antibodies used for western blotting and immunohistochemistry were as follows: anti-Bcl-2 (Boster, China), anti-Bax (Boster, China), anti-IκBα (Bioss, China), anti-cleaved caspase-3 (Abcam, USA), anti-NF-κB (Boster, China), anti-p-NF-κB (Bioss, China), anti-Cox-2 (Boster, China), anti-iNOS (Bioss, China), anti-β-actin (Boster, China), and anti-Histone H3 (Bioss, China).