Western and ip buffer

Cells were harvested and lysed in Western and IP buffer (Beyotime Biotechnology, Haimen, China), and total protein concentrations were determined using the BCA Protein Assay Kit (Beyotime Biotechnology, Haimen, China). Equal amounts of cell lysates were loaded and separated on a 12% SDS-PAGE gel and transferred to 0.22-μm PVDF membranes (Millipore Billerica, MA, USA). The membranes were blocked with QuickBlock™ Blocking Buffer (Beyotime Biotechnology, Haimen, China) for 15 min at room temperature and then incubated with primary antibodies overnight at 4 °C. Then, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature.
The following primary antibodies were used for blotting. Mouse antibodies against p21, p16, p53, Rb, Runx2, PPARγ, γ-H2AX, CD63, Tsg101, and CD81 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse antibodies against β-actin and rabbit antibodies against β-catenin were purchased from Beyotime Biotechnology (Beyotime Biotechnology, Haimen, China). Rabbit antibodies against calnexin, SOD1, SOD2, and catalase were purchased from Abcam (Abcam, Cambridge, UK). The specific information of antibodies is listed in Table S1.

The BmN-SWU1 cells (2 × 10⁵) were plated in 24-well plates, and transfected with 0.8 μg plasmid. At the indicated time points, cells were harvested for Western blotting. The cell samples were lysed using Western and IP buffer [20mM Tris(pH7.5), 150 mM NaCl, 1% Triton X-100] (Beyotime, China). The total protein concentration was determined using a BCA Protein Assay Kit (Beyotime, China). After SDS-PAGE, the proteins were transferred onto a nitrocellulose membrane (Roche, Switzerland) and incubated with indicated primary antibodies, respectively. Then, the membrane was further incubated with HRP-labeled secondary antibodies. The blots were visualized using a Clarity Western ECL Substrate (Bio-Rad, USA).

Co-IP was performed as previously described [47 (link)]. In brief, the MM cell lines MM.1S and RPMI 8226 were treated with 10 nM of NNU219 for 24 h. The control group was incubated in the same concentration of DMSO in culture medium. The stimulated cells were lysed in western and IP buffer (Beyotime, Jiangsu, China) and centrifuged at 4°C. Correlative proteins were immunoprecipitated from 1 mg MM cell lysate using IκBα (1:1000) or IgG (1:1000) antibody (rabbit) and coupled to protein A/G agarose beads (Beyotime, Jiangsu, China) overnight at 4 °C. The beads were washed with IP buffer, and the elution was processed by ubiquitin or IκBα immunoblotting.

Cells were lysed in Western and IP buffer (Beyotime, China) containing protease inhibitor cocktail (Thermo scientific). The whole cell lysates (WCL) were pre-cleared by incubating with 1.0 μg of control IgG corresponding to the host species of primary antibody, together with 20 μl Protein A/G PLUS-Agarose (Santa Cruz) at 4 °C for 2 h, followed by centrifugation at 4°C for 5 min at 2, 500 rpm. The supernatant was subjected to immunoprecipitation by addition of 2 μg immunoprecipitation antibodies and incubated overnight at 4 °C followed by incubation with Protein A/G PLUS-Agarose for 2 h. After washing with cell lysis buffer, the beads were boiled in 2×SDS loading buffer and the supernatants were resolved by SDS-PAGE. For the GST-pulldown assays, GST-GRP78 or GST immobilized on Sepharose 4B-glutathione beads (GE Healthcare) were incubated with pre-cleared WCL at 4 °C for 12 h followed by washing with the lysis buffer. The bound proteins were dissolved in 2×SDS loading buffer, separated by 10% SDS-PAGE, and immunoblotted with the APC antibody.

Cells were lysed in Western and IP buffer (Beyotime, China) containing a protease inhibitor cocktail (Thermo scientific). 800 μg supernatant from whole-cell lysates (a final volume of 1 ml) were pre-incubation with 1.5 μg control IgG corresponding to the host species of the primary antibody and immunoprecipitation antibodies overnight followed by incubation with 50 μl of Protein A/G PLUS-Agarose (Santa Cruz) at 4° C for 2 h. Beads were isolated by centrifugation at 4° C for 5 min at 2,500 rpm. After three times washing with cell lysis buffer, beads were boiled in 2 × SDS loading buffer for 5 min, and supernatants were loaded on SDS-PAGE and subjected to Western blot analysis, as previously described.

For all samples, cells were lysed on ice for 30 min with Western and IP buffer (Beyotime, China) and cytoplasmic proteins in supernatant were collected by centrifugation at 10,000 g for 10 min. Subsequently, cytoplasmic proteins were separated by 12% SDS–PAGE gel and transferred to PVDF membranes (Millipore, United States). Membranes were blocked in TBS containing 5% BSA and 0.05% Tween-20 for 1 h at room temperature. Incubations with diluted primary antibodies in TBS 0.05% Tween-20 were performed at 4°C overnight. After 2 h incubation with an anti-rabbit or anti-mouse peroxidase-conjugated antibody (1/5000, Sigma, United States) at room temperature, proteins were detected by chemiluminescence (Thermo Fisher Scientific, United States). These antibodies were used: rabbit anti-NK-κB1 p105 (1/1000, Cell Signaling, United States), rabbit anti-phospho-IκBα/β (1/1000, Bioss Antibodies, China), mouse anti-NF-κB p52 and mouse anti-β-actin (1/1000, Santa Cruz, United States).

HEK293T cells were co-transfected with pcDNA-HA-CaMKIIα and pCAGGS-Flag-NS3 or pCAGGS-Flag-NS5 using ProFection calcium phosphate reagents (Promega). Subsequently, 24–36 h post-transfection, the cells were collected using western and IP buffer (Beyotime, Jiangsu, China) containing PMSF protease inhibitor according to the manufacturer’s instructions. Whole-cell extract (30 μL) was used as input; the remainder was divided into two equal parts and incubated with the Flag antibody and IgG, separately, at 4°C overnight with gentle rotation. Protein G magnetic beads (Invitrogen) were added into the lysates and incubated for 2–3 h according to the manufacturers’ instructions. Precipitates were washed five times with TBST buffer, and the proteins were eluted by boiling the beads for 5 min in 1× SDS sample buffer. Eluted proteins and input whole-cell extracts were analyzed by western blot.