Sc 53909

Antibodies against phospho-ERK1/2 (4370), phospho-p38 (9215), phospho-JNK1/2 (9251), phospho-IκBα (9246), TAK1 (4505), TAB2 (Leu 330, 3745), SUMO1 (4930), and HA-tag (3724) were obtained from Cell Signaling Technology. Antibodies against ERK1/2 (201245-4A4), p38 (200782), JNK1/2 (201001), IκBα (200517) and TRAF6 (380803) were obtained from Zen Bioscience, China. Anti-β-Actin (A2228), anti-Flag (F1804), and HRP-conjugated anti-Flag (A8592) antibodies were purchased from Sigma-Aldrich. Anti-Myc (MA1-21316), HRP-conjugated anti-Myc (MA1-81357), and HRP-conjugated goat anti-rabbit IgG (31460) antibodies were purchased from Invitrogen. Antibodies against TRAF6 (SC-8409), TAB2 (N48-73, SC-398188), and GST (SC-53909) were obtained from Santa Cruz Biotechnology. The antibodies against RIP1 (610458), ubiquitin (04-263), and K63-linked ubiquitin (BML-PW0600) were purchased from BD Bioscience, Merck Millipore, and Enzo Life Science, respectively. The HRP-conjugated anti-mouse secondary antibody (KCB002) was purchased from Rockland.
LPS (E. coli O127:B8, L3129), MG132 (474790), polybrene (H9268) and chloroquine (C6628) were purchased from Sigma-Aldrich. CpG-ODN 1826 (tlrl-1826) and puromycin (540411) were obtained from InvivoGen and Merck Millipore, respectively. Murine TNFα (AF-315-01A) was purchased from PeproTech.

The GST-tagged pGEX-6P-1 vector (GE Healthcare Life Sciences) was used for sub-cloning TKTKK1 by fusing with the GST sequence at its 3′-terminal. After verification by sequencing, the new plasmid pGEX-6P-1 with GST-TKTKK1 was transformed into the E. coli BL21. A total of 500 mg of rice seed power in each sample was used for protein extraction and the resulted supernatant was transferred into SnakeSkin^TM Dialysis Tubing (10K MWCO, 22 mm, ThermoFisher Scientific) for dialysis against PBS buffer (change buffer 8–12 hours) at the chill room for 2 days. Crude proteins were separated on the mini-protein precast gel (Bio-Rad) and were then transferred onto nitrocellulose membrane.
For detecting protein expression in the E. coli system, GST (1E5) mouse monoclonal, SC-53909 from Santa Cruz Biotechnology was used as the primary antibody. The anti-mouse IgG HRP from GE Healthcare Life Sciences was used as the secondary antibody. For detecting the protein expression in the rice seeds, the 14-aa peptide KKKTKTKTRSTKTK specific to the synthetic genes was used as antigen for antibody synthesis by GenScript, Piscataway, NJ. The HRP- Goat-Rabbit IgG (H+L) DS Grd (from Life technologies) was used as the secondary antibody. Western blot hybridization was carried out using Bio-Rad's Western blotting systems according to the manufacturer’s instructions.