Iblot gel transfer stacks nitrocellulose mini kit

Cells were washed once in PBS and lysed in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, and 2 mM EDTA) containing phosphatase and protease inhibitors (100 mM sodium fluoride, 1 mM sodium orthovanadate, 40 mM β-glycerophosphate, 10 µg/mL leupeptin, 1 µM pepstatin A, and 1 mM phenylmethylsulfonyl fluoride). Protein extracts were separated by NuPAGE^® Bis-Tris gels (Thermo Fisher Scientific) and dry blotting was performed using iBlot^® Gel Transfer stacks Nitrocellulose Mini kit and iBlot^® machine (Invitrogen). Primary human antibodies for hCAP-18 (#650302) and C/EBPα (#662102) were purchased from Biolegend. Household β-actin antibody (A1978) was purchased from SIGMA-ALDRICH and used as a loading control. Secondary antibodies (IRDye^® 800CW Goat anti-Mouse, IRDye^® 680RD Goat anti-Mouse) were purchased from LI-COR Biosciences. LICOR Odyssey imager was used as the scanning system.

Following treatment cells were washed once in PBS and lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 0,5% Triton X-100 and 2 mM EDTA) containing phosphatase and protease inhibitors (100 mM Sodium Fluoride, 1 mM Sodium Orthovanadate, 40 mM β-Glycerophosphate, 10 µg/mL Leupeptin, 1 µM Pepstatin A and 1 mM Phenylmethylsulfonyl fluoride). Protein extracts were separated by NuPAGE® Bis-Tris gels (Thermo Fisher Scientific) and dry blotting was performed using iBlot® Gel Transfer stacks Nitrocellulose Mini kit and iBlot® machine (Invitrogen). Primary human antibody (mouse) for phospho-NF-κB p65 (sc-136548) was purchased from Santa Cruz. Primary human antibodies (rabbit) for NF-κB p65 (#3034), phospho-IRF3 (#4961) and IRF3 (#4962) were purchased from Cell Signaling Technology. Household β-actin antibody (mouse) (A1978) was purchased from SIGMA-ALDRICH and used as a loading control. Secondary antibodies (IRDye® 800CW Goat anti-Mouse, IRDye® 800CW Goat anti-Rabbit, IRDye® 680RD Goat anti-Mouse) were purchased from LI-COR Biosciences. LICOR Odyssey imager was used as the scanning system and relative intensity of protein bands was quantified with ImageJ.

After extraction, the histones (500ng) were separated on 4–20% Tris-Glycine gels (Invitrogen) and transferred onto a nitrocellulose membrane (0.2 mm) using the iBlot^® Gel Transfer Stacks Nitrocellulose kit Mini (Invitrogen). After blocking nonspecific sites for 1 h at room temperature in a solution of 1X TBS at 5% milk, the membrane was incubated with rabbit anti-H3K79me2 (ab3594, 1/1000) or anti-H3 (ab1791)1/1000) overnight at 4 °C or 1 h at RT. The membrane was then washed three times for 5 min in a 1X TBS solution containing 0.05% Tween20 and was then incubated with an HRP anti-rabbit secondary antibody (NA934V, 1/10,000) for 1 h at RT. After 3 washes of the secondary antibody, the membrane was revealed using the ECL™ Prime Western Blotting Detection Reagents Developer Kit (GE Healthcare). The PageRuler prestained protein ladder 10 to 180 kDa (Fisher Scientific) was used as a molecular weight marker. The antibody-labeling of histone H3 was used as an internal control for loading and amount of histones in each well. At least three independent experiments were run.