Anti gst

H9C2 cells with different treatment were lysed, quantified and subjected to SDS-PAGE electrophoresis. Proteins were transferred to PVDF membranes (Cat # 1,620,177, Bio-Rad, Hercules, CA, USA) which were incubated with 5% nonfat milk in TBST and followed by incubation of primary antibodies at 4°C overnight. Then the PVDF membranes were washed with TBST and incubated with secondary antibodies. Protein expression was detected using BeyoECL Moon (Cat # P0018FS, Beyotime) under a Tanon 5200 machine (Shanghai, China). The detailed primary and secondary antibodies were listed as below: anti-cleaved caspase 3 (Cat # E83-77, Abcam); anti-caspase 3 (Cat # ab32351, Abcam); anti-Bax (Cat # WL01637, Wanleibio, Shenyang, China); anti-Bcl-2 (Cat # WL01556, Wanleibio); anti-β-actin (Cat # WL01372, Wanleibio); anti-Nrf2 (Cat # WL02135, Wanleibio); anti-Sirt2 (Cat # ab211033, Abcam); anti-HO-1 (Cat # EP1391Y, Abcam); anti-GST (Cat # ab138491, Abcam); anti-NQO1 (Cat # 11,451-1-AP, Proteintech, Wuhan, China); anti-FOXO3a (Cat # 66,428-1-Ig, Proteintech); anti-GCLM (Cat # 14,241-1-AP, Proteintech); anti-Keap1 (Cat # 10,503-2-AP, Proteintech). IgG(H + L)(HRP-labeled Goat Anti-Rabbit IgG(H + L)) and IgG(H + L)(HRP-labeled Goat Anti-Mouse IgG(H + L)) were purchased from Beyotime (Beijing, China). All protein expressions were normalized by β-actin expression.

The coding sequence of TLR3 was cloned into the pGEX4T-1 vector to facilitate the production of recombinant GST-tagged protein. Similarly, the coding sequences of Cycb3, NFY1 and 7721 were subcloned into the pET32a vector to generate recombinant His-tagged proteins. All resulting clones were transformed into Escherichia coli strain BL21 and the production of the recombinant protein was induced at 18 °C for 12 h by adding 0.5 mM IPTG. The GST-tagged protein and His-tagged proteins were subsequently purified using Ni–NTA agarose beads (QIAGEN) and glutathione Sepharose beads (Amersham Biosciences), respectively. The GST-tagged proteins and His-tagged proteins were incubated at 4 °C for 3 h in binding buffer (300 mM NaCl, 20 mM Tris–HCl, 1% [v/v] Triton X-100, Cocktail [Roche, Code No. 4906845001]). The bound proteins were separated by SDS-PAGE, transferred to a PVDF membrane (Millipore Code No. IPFL00010), and detected by immunoblot analysis with anti-His (Abmart,1:5,000) and anti-GST (Abcam,1:5,000) antibodies.

COS cells were plated at cell density of 8 × 10⁵/10 cm on tissue culture plates. Cells were transfected using GeneJuice (Merck Millipore, Darmstadt, Germany) as indicated with 5 μg of Myc-CIITA, Flag-pCAF, Flag-K141R, K144R, K141/144R CIITA, HA-K48 Ub, K63 Ub, HA-mono Ub, or pCDNA control. Twenty-four hours after transfections, cells were lysed in 1% NP40 buffer supplemented with EDTA-free protease inhibitors (Roche) on ice. Lysates were centrifuged, normalized for protein concentration, and precleared with Mouse IgG (Sigma-Aldrich) and Protein G (Thermo Fisher) followed by immunoprecipitation with either EZ view anti-c Myc affinity gel beads (Sigma-Aldrich) or with anti-Flag M2 affinity gel (Sigma-Aldrich). Immune complexes were denatured with Laemmli buffer, boiled, and separated by SDS-PAGE gel electrophoresis. Gels were transferred to nitrocellulose and were individually immunoblotted with anti-Myc (Abcam, Cambridge, MA), anti-Flag (Sigma-Aldrich), antiubiquitin (Life Sensors, Malvern, PA), anti-K48 ubiquitin (Cell Signaling, Danvers, MA), anti-K63 ubiquitin (Millipore), or with anti-GST (Abcam, Cambridge, MA). HRP conjugates were detected using HyGlo Chemiluminescent substrate (Denville). Protein normalization and equal loading were determined in lysates.

The GST-pull down assay was performed as described previously [34 (link)]. Purified His-LcNAC13 was incubated with GST or GST-LcR1MYB1 bound to glutathione Sepharose 4B beads (GE Healthcare, Pittsburgh, PA, USA). The eluted proteins were subjected to SDS-PAGE and Western blotting. Gel blots were analyzed using anti-His antibody and anti-GST (Abcam, Cambridge, MA, USA) antibodies. The chemiluminescent signal was visualized with enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA).

Antibodies used were: anti-SETD6 (Genetex, GTX629891), anti-Plk1 (Millipore, 05-844), anti-pan-methyl (Abcam, ab23366), anti-FLAG M2 (Sigma, F1804), anti-GAPDH (Abcam, G041) and anti-GST (Abcam, ab9085). HRP-conjugated secondary antibodies (goat anti-rabbit and goat anti-mouse) were purchased from Jackson ImmunoResearch (111-035-144 and 115-035-062, respectively).