Chemiscope 3000 mini

Immunoprecipitation, SDS-PAGE and immunoblot analysis were performed according to a standard protocol as described previously (35 (link)). The following antibodies were used according to the manufacturers’ protocols: anti-Fbxw7 (Abcam, ab12292, RRID: AB_442966), anti-Lys48-specific linked polyubiquitin (MilliporeSigma, 05-1307, RRID: AB_1587578), anti-c-Jun (CST, #9165S, RRID: AB_2130165), anti-p65 (CST, #8242, RRID: AB_10859369), anti-p-p65 (CST, #3036, RRID: AB_331281), Collagen-I(Abcam, ab34710, RRID: AB_731684), Collagen-III (Abcam, ab7778, RRID: AB_306066), EGR-1 (CST, #4154S, RRID: AB_2097035). The PVDF membranes were incubated with corresponding primary antibodies followed by horseradish peroxidase-linked secondary antibodies. The images were developed and captured using Chemiluminescence imaging system (ChemiScope 3000 Mini, Clinx Science Instruments Co., Ltd). Relative quantities of target protein were determined comparing to β-actin expression using densitometric analysis (ImageJ 1.52v). The standard deviation was calculated for biological duplicates.

Samples were spun and cell pellets were re-suspended in 1×SDS loading buffer (3% SDS, 10% glycerol, 50 mM Tris–HCl pH 6.8, 0.1% bromophenol blue, 12.5 mM EDTA, 100 mM DTT) to a concentration of approximately 10⁶ cells/μl and boiled at 95°C for 10 min. Protein concentration was measured according to the manufacturer’s instructions for the BCA Protein Assay Kit (Solarbio, Beijing, China). Total proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes (Merck Millipore, USA). GFP fusion proteins and RNA polymerase subunit α (RNAPα) were detected using antibodies directed against GFP (1:2000; mouse; Roche), anti-E.coli RNAP (1:2000; mouse; BioLegend, San Diego, CA, USA), and goat anti-mouse secondary antibodies conjugated with horseradish peroxidase (1:5000; Transgen Biotech). Signals were visualized using Western Lightning Plus-ECL (Edo Biotech) and detected with ChemiScope 3000 mini (CLiNX, Shanghai, China).

Total RNA was purified from Xoc BLS256 liquid cultures (OD₆₀₀ = 1.0) using the EasyPure RNA Kit (Transgen Biotech, Beijing, China). RNA (10–20 μg) was separated in 1% agarose gels containing 25 mM guanidium thiocyanate, transferred to Hybond N+ nitrocellulose membranes (Merck Millipore, USA), and cross-linked to membranes by UV radiation. Probes were 5’-labeled with digoxygenin (DIG). Membranes were prehybridized for 10 min at 42°C, and then incubated with labeled probes overnight. Membranes were then rinsed, dried and visualized by phosphorimaging on a ChemiScope 3000 mini (CLiNX, Shanghai, China) as described previously [13 (link)].

The Hfq and Xoc_3982 proteins were expressed and purified using the intein-based Impact Kit (New England Biolabs, USA) as described [9 (link)]. Binding reactions were conducted in 10 μl volumes with the LightShift Chemiluminescent RNA EMSA Kit (ThermoFisher, USA); reactions were incubated at 37°C for 20 min, and 5 μl of loading buffer (50% glycerol) was then added. The interaction of Hfq and Xonc3711 sRNA was conducted in 1× binding buffer with 3’-biotinylated Xonc3711 sRNA. The interaction of sRNA Xonc3711-with Xoc_3982 mRNA was investigated using EMSA as described previously [9 (link)]. Samples were separated in 5% nondenaturing polyacrylamide gels in 0.5× TBE at 4°C and visualized by phosphoimaging on a ChemiScope 3000 mini (CLiNX, Shanghai, China).

In brief, protein samples, which were extracted from Huh-7 or SR cells, were standardized using a BCA protein assay kit, loaded onto 8–12% SDS-PAGE, transferred to a PVDF membrane, and blocked with Tween-Tris-buffered saline (TBST) solution supplemented with 5% BSA. Subsequently, the membrane was incubated with a primary antibody at 4°C overnight. The next day, after washing with TBST, these membranes were incubated with a secondary antibody for 2 hours at room temperature, followed by enhanced chemiluminescence. Blotting was visualized using chemiluminescence (ChemiScope 3000 mini, Clinx Science Instruments Co., Ltd., Shanghai, China) following the manufacturer's instructions. β-Actin was selected as an internal control to compare protein levels. The intensity of the bands was determined based on Image J software.

RAW264.7 cells (1 × 10⁶/well) were cultured in a 6-cell plate overnight and then pretreated with E9OAEE (6.25, 12.5, 25, 50 µg/mL) for 2 h and stimulated with LPS (1 µg/mL) for 24 h or indicated time, respectively. Cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China) on ice for 30 min, and the supernatant was collected. Proteins were quantified using Enhanced BCA Protein Assay Kit (Beyotime, Jiangsu, China), and equal amounts of protein (40 µg) were separated via 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and then transferred onto PVDF membranes (Millipore, CA, USA). The membranes were blocked with 5% skim milk at room temperature for 1 h and then incubated overnight at 4°C with primary antibodies specific for iNOS (ABclonal, Wuhan, China), GAPDH (Bioworld Technology, Inc, MN, USA), COX2 (Bioworld Technology, Inc, MN, USA), JNK1/2/3 (ABclonal, Wuhan, China), p-JNK (Bioworld Technology, Inc, MN, USA), P38 (ABclonal, Wuhan, China), p-P38 (Bioworld Technology, Inc, MN, USA), ERK1/2 (ABclonal, Wuhan, China), and p-ERK1/2 (ABclonal, Wuhan, China). The membrane was then incubated for an additional 60 min with a goat anti-rabbit lgG/HRP (Bioss, Beiing, China). Then, the membrane was developed using Super ECL Plus kit (US Everbright Inc, Suzhou, China) for imaging with ChemiScope 3000 mini (Clinx, Shanghai, China).