Trans blot sd cell

Proteins separated by SDS-PAGE were transferred to polyvinylidene difluoride (PVDF) membranes (MERCK, Tokyo, Japan) for 90 min at 200 mA constant current using a Trans-Blot SD cell (BIO-RAD, California, America). Immunostaining was performed using rabbit anti-human polyclonal antiserum (MBL, Nagoya, Japan), for primary immunoreaction, and Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H + L) serum (MBL) for secondary immunoreaction. After washing the membranes with PBS-T (pH 7.4), bound antibodies were detected with POD Immunostain Set (FUJIFILM Wako, Osaka, Japan).

PI3K, Akt and p-Akt, JAK, STAT3, and pSTAT3 protein expressions were measured using western blot (WB). All antibodies for Western blot analysis were purchased from Cell Signaling Technology (Beverly, MA). β-actin (13E5) Rabbit mAb (CST#4970), PI3 kinase p85 (19H8) Rabbit mAb (CST#4257), Akt antibody (CST#9272), p-Akt antibody (Ser473) (D9E) XP^® Rabbit mAb (CST#4060), JAK3 (D7B12) Rabbit mAb (CST#8863), STAT3 (D3Z2G) Rabbit mAb (CST#12640), phospho-Stat3 (Tyr705) (D3A7) XP^® Rabbit mAb (CST#9145), Anti Rabbit IgG HRP Link-Antibody. The sample used in testing was 70 μg of protein. SDS-page electrophoresis was done using a buffer tank Electrophoresis Apparatus Vertical TV100Y (Scie-Plas). Proteins that have been successfully separated will be transferred using semi-dry Trans Blot-SD Cell (Bio-Rad). The next stage is blocking by using skim milk and BSA. After blocking, the membrane is incubated with primary antibodies in a ratio of 1: 1,000. Incubation was carried out in Cold Room 4°C (Fiocetti) for 16–18 h. After incubation with a primary antibody, the membrane is washed and then incubated with secondary antibodies 1: 5,000. The target protein band on the membrane was detected using the Gel Documentation System with Chemiluminescence Alliance 4.7 (Uvitec) with enhanced chemiluminescence (ECL) substrate, Clarity Western.

Followed by electrophoresis, proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, United States) using Trans-Blot^® SD Cell (Bio-Rad, United States). The virus overlay assays were applied according to the methods reported by Wu et al. (2007) (link) and Cheng et al. (2014b) (link) with some modifications. Briefly, proteins on the membrane were denatured in high concentration the guanidine-HCl and then renatured in gradually reducing the guanidine-HCl concentration. The renatured proteins were subsequently incubated overnight with BmNPV particles. After washed three times in PBST, the membranes were incubated with monoclonal antibodies (MAbs) against Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) gp64 protein (1:500, Santa Cruz). After washing three times, antigen-antibody complexes were detected with goat anti-mouse secondary antibody (1:5000, TransGen biotech, China). After washing three times, the membranes were visualized using a diaminobenzidine (DAB) kit (Tiangen, China) according to the manufacturer’s instructions. For the negative control, the membranes were incubated without BmNPV particles, other steps were all the same. The parallel SDS-PAGE gels and 2-DE gels stained with Coomassie brilliant blue R-250 were used to locate the potential virus-binding proteins.

Protein was extracted from WAT with RIPA buffer and quantified using the BCA method. Then, 20 μg of protein was separated by 8–10% SDS-PAGE at 100 V for 90 min. Separated proteins were transferred to PVDF membranes by semi-dry transfer cells (Trans-Blot SD Cell; Bio-Rad, Hercules, CA, USA) at 15 V for 30 min. Membranes were blocked with 5% skim milk in TBST for 1 h at room temperature. The membrane was washed 3 times with TBST and incubated overnight at 4 °C with the primary antibody. Afterward, the membrane was rewashed 3 times and incubated with the secondary antibody for 2 h at room temperature. After the incubation, the membrane was washed 3 times, and the specific protein bands were chemiluminescent by ECL solution and visualized with a Western blotting detection system (ImageQuan LAS 500; GE Healthcare, Chicago, IL, USA). The shaded areas of specific bands were quantified using Image J software.

Total proteins from all the cell samples were extracted using RIPA buffer containing 1 mM PMSF (Beyotime, Haimen, China). 50 μg of each protein was loaded on 10% SDS polyacrylamide gels for electrophoresis, and then the proteins were transferred to PVDF membranes (Millipore, USA) by a Transblot SD Cell semidry transfer machine (Bio Rad, USA). After being blocked by 5% nonfat milk, the membranes were incubated with goat anti-PinX1 antibody (1 : 500, Santa Cruz Biotechnology, Santa Cruz, USA) and HRP conjugated rabbit anti-goat IgG (1 : 5000, Multisciences, Hangzhou, China) for the detection of PinX1 protein. β-actin was detected by using the mouse anti-β-actin antibody (1 : 2000, Multisciences, Hangzhou, China) and HRP conjugated goat anti-mouse IgG antibody (1 : 5000, Multisciences, Hangzhou, China). HRPs on the immune complex were visualized by the BeyoECL Plus Kit (Beyotime, Haimen, China) and images were taken by the Image Station 4000R PRO scanner (Carestream Health, USA).

To validate the specificity of the newly developed antibodies, we performed a western blot analysis, as follows. The purified recombinant proteins ATXβ and ATXδ and serum samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to a polyvinylidene fluoride membrane using a semi-dry blotter (Transblot SD Cell, Bio-Rad). The membranes were blocked for 2 hours at room temperature in Tris-buffered saline containing 3% powdered milk, then incubated with the anti-classical ATX antibody R4+4 (1 μg/mL) or the anti-novel ATX antibody R4-127 (1 μg/mL). After reaction with alkaline phosphatase-labeled anti-rat IgG, the monoclonal antibodies that were bound to the ATX isoforms were visualized using an enhanced chemiluminescence kit (CDP-STAR, Roche).