Ecl substrate kit

The HeLa cells were seeded overnight before transfection with mRFP tagged IN. The cells were harvested after 24 h and lysis was done in 0.25% Nonidet (P-40) buffer. The supernatant (500 µg/mL) were collected after centrifugation at 12,000g for 30 min at 4 °C. It is then treated with MNase enzyme. The protein quality was analysed on SDS PAGE gel. The nuclear protein were then incubated with anti-IN antibody bound protein A/G agarose beads for 4 h at 4 °C with gentle rocking. The complex was eluted and subjected to SDS–PAGE gel electrophoresis as described earlier [40 (link)]. The proteins were then transferred to nitrocellulose membrane (MDI), treated with anti IN and anti-PSF antibody. HRP conjugate anti-mouse antibody was used as secondary antibody. The visualization of protein was done by ECL substrate kit (Thermofischer).

Nuclear extracts from U87 and U251 cells were prepared in accordance with the manufacturer's protocol (NE-PER® Nuclear and Cytoplasmic Extraction Reagents, Pierce). Complementary oligonucleotide pairs corresponding to the E-box 2 and E-box 3 embedded in the promoter region of MC-let-7a-1~let-7d and 5′ end-labeled with biotin by Invitrogen. The oligonucleotide sequences were shown in Supplementary Table S2. EMSA assay was performed by using a LightShift^® Chemiluminescent EMSA Kit (Pierce). Binding reaction with 10 μg of HepG2 nuclear extracts and 100 fmol 5′ Biotin-labeled oligonucleotide was carried out in accordance with the manufacturer's instructions. For the competition assay, a 100-fold molar excess of unlabeled oligonucleotide was added to the binding reaction mixture as a specific competitor. For antibody-supershift assay, a nuclear extract was pre-incubated with a 3 μl MYC antibody (Abcam) before adding it to the binding reaction. DNA/protein complexes were separated on a pre-electrophoresed 6% polyacrylamide gel in 0.5 × TBE, transferred to a nylon membrane, and cross-linked at 120 mJ/cm² for 1 minute and detected by chemiluminescence, in accordance with the manufacturer's directions. Membranes were exposed by using the enhanced chemiluminescence (ECL) substrate kit (Thermo) and then were photo-documented.

Total proteins were extracted from N. benthamiana plant leaves in extraction buffer (10% glycerol, 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM DTT, 2% polyvinylpolypyrrolidone, 1× protease inhibitor cocktail, 0.2% TritonX-100). The crude plant extracts were centrifuged at 12,600× g for 10 min, and the supernatant was mixed with 3 × SDS loading buffer (150 mM Tris-HCl, pH 6.8, 6% SDS, 0.3% bromophenol blue, 30% glycerol, 300 mM DTT) and boiled for 10 min. Protein samples were separated in 10% SDS-PAGE gels and then transferred to PVDF membranes. The blots were probed with an anti-TSWV N antibody followed by HRP-conjugated goat anti-rabbit antibodies (1:10,000). An ECL Substrate Kit (Thermo Scientific, Hudson, NH, USA) was used to develop the blots. A Bio-Rad ChemiDoc Touch imaging system (Bio-Rad, Hercules, CA, USA) was used to visualize the signals.

CAR-T cells were incubated with 2 µg anti-FLAG Ab in 100 µL PBS for 20 mins on ice and then with 2 µg goat antimouse secondary Ab for another 20 mins on ice. Cells were then incubated in the 37°C water bath for the selected time points and then lysed with 2 x Laemelli buffer for 10 mins. Cell lysates were then separated in 4% to 15% 10 well SDS-PAGE gels and transferred to polyvinylidene difluoride membranes at 75V for 120 mins (Bio-Rad). Blots were examined for human CD3ζ (Santa Cruz Biotechnology), p-Y142 CD3ζ (Abcam), pan-ERK (BD Biosciences), and pan-Akt, p-S473 Akt, and p-T202/Y204 MAPK (Cell Signaling Technology) with 1:1000 dilution in 5% TBS-Tween milk. Membranes were incubated with HRP-conjugated secondary goat anti-mouse or goat anti-rabbit IgG (Santa Cruz) at a dilution of 1:3000 and imaged with the ECL substrate kit (Thermofisher) on the ChemiDoc MP System (Bio-Rad) according to the manufacturer’s instructions.26 (link)

Cultured HaCaT cells were incubated in serum-free DMEM for 24 h and then treated with 10, 20, or 40 μM of lotusine for 1 h, followed by sUV irradiation. For MMP-1, the medium was harvested on ice and centrifuged at 15,000× g for 15 min. For protein extraction, cell lysates were prepared using cell lysis buffer (50 mM Tris-HCl at pH 8.0, containing 0.15 M NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1mM sodium vanadate). The protein concentration was determined using a protein analysis kit (Bio-Rad, Hercules, CA, USA). Proteins were electrophoretically separated using a 10% SDS-polyacrylamide gel and subsequently transferred onto polyvinylidene fluoride membranes (Merck Millipore, Burlington, MA, USA). After blotting, the membranes were blocked with 5% skim milk in 10% phosphate-buffered saline for 1 h. The membranes were incubated overnight with specific primary antibodies at 4 °C. The protein bands were visualized using an ECL substrate kit (Thermo Fisher Scientific, Waltham, MA, USA) after the addition of horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz, CA, USA). Western blot data were analyzed using quantitative analysis in the Image Studio software (LI-COR, Lincoln, NE, USA).

Total protein was extracted from cultured cells by using RIPA buffer supplemented with 1% proteinase inhibitor and 1% phosphotransferase inhibitor (Boster Biotechnology). Proteins were separated by 10% SDS‐PAGE gel electrophoresis and then transferred to PVDF membranes (Millipore). The membranes were blocked with 5% BSA a for 1 hour and then incubated with primary antibodies followed by washing with Tris‐buffered saline Tween 20 (TBST) for three times. Blots were then incubated with horseradish peroxidase–conjugated secondary antibodies, and the signals were detected with ECL Substrate Kit (Thermo Scientific).