Ta503129

Plasmids encoding Myc-H1.2/H1.3/H1.4/H1.5/H1t were synthesized by Genscript Co., Ltd. (Nanjing, China). The expression plasmids Flag-MDA5/MAVS/TBK1/IRF3 (5D) (the active mutant of IRF3) were all constructed and stored by our lab.
Polyclonal antibodies anti-Flag tag (20543-1-AP), anti-Myc-tag (60003-2-Ig), anti-IFIH1/MDA5 (21775-1-AP), anti-MAVS (14341-1-AP), anti-TBK1 (28397-1-AP), anti-IRF3 (11312-1-AP), and anti-HSP90 (60318-1-Ig) were purchased from Proteintech Biotechnology (Wuhan, China). Mouse monoclonal antibody for β-tubulin (TA503129) was bought from OriGene Technologies, Inc. Anti-Phospho-IRF3 (S386) recombinant rabbit monoclonal antibody (SU03-28) and monoclonal antibodies of anti-Myc tag (EM31105), H3 (M1309-1) and H1.2 (ET1706-26), were purchased from Huabio (Hangzhou, China). Peroxidase- and Cy3-Labeled antibodies of Goat Anti-Mouse IgG (H+L) and 488-Labeled Goat Anti-Rabbit IgG (H+L) were bought from Jackson ImmunoResearch Inc. (West Grove, PA, USA).

Proteins were collected, quantified with a BCA kit, and boiled samples (20 μg) were separated via SDS-PAGE, then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 2 h and then incubated with primary antibodies, including anti-p-STAT6 (ab263947, Abcam, Cambridge, UK, 1:1000), anti-STAT6 (ab32520, Abcam, 1:1000), anti-p-JAK2 (ab32101, Abcam, 1:1000), anti-JAK2 (ab108596, Abcam, 1:1000), anti-p-STAT3 (ab76315, Abcam, 1:1000), anti-STAT3 (ab68153, Abcam, 1:1000), anti-Bcl-2 (ab32124, Abcam, 1:1000) and anti-β-tubulin (TA503129, OriGene, Rockville, MD, USA, 1:1000) at 4 ℃ overnight. Next, the membrane was incubated with an HRP-conjugated secondary antibodies for 1 h and proteins were visualized using the ECL Western Blotting Detection System (ChemiDoc XRS, Bio-Rad, Hercules, CA, USA).