Polyvinylidine difluoride membrane

Cultured cells lysed in mammalian lysis buffer (Sigma-Aldrich), with appropriate phosphatase and protease inhibitors, followed by scraping. Tissues were homogenized by MagNA Lyser beads (Roche) in the same buffer. Protein concentration was determined using the BCA Kit (Bio-Rad), and protein was stored at −80 °C. Prior to western blotting, Laemmli sample buffer was added to the samples (250 mmol/l Tris, pH 7.4, 2% w/v SDS, 25% v/v glycerol, 10% v/v 2-mercaptoethanol, and 0.01% w/v bromophenol blue), and the samples were then heated to 105 °C for 5 minutes, kept at 4 °C for 10 minutes, followed by separation on a SDS-polyacrylamide gel. Proteins were then transferred to a polyvinylidine difluoride membrane (Bio-Rad) in transfer buffer (25 mmol/l Tris, pH 8.8, 192 mmol/l glycine, and 10% v/v methanol). All washing, blocking and antibody solutions were prepared in PBS with 0.1% Tween-20 (PBST). Membranes were blocked for one hour in 5% milk, followed by overnight incubation at 4 °C with primary antibodies in 1% bovine serum albumin. Membranes were washed thrice, followed by secondary antibody incubation at room temperature for 1 hr. in 1% bovine serum albumin, followed by 3 more washes in PBST, and placed in PBS. Blots were probed using an enhanced chemiluminescence system (GE Healthcare) on a GelDoc imager (Bio-Rad). Densitometry was performed using ImageJ software (NIH).

Cultured cells were lysed in mammalian lysis buffer (Sigma-Aldrich) while tissue samples were homogenized by MagNA Lyser (Roche) in the same buffer. Concentration of protein was determined by the BCA Kit (Bio-Rad). Protein was stored at −80°C. Immediately prior to western blotting, Laemmli sample buffer was added (250 mmol/l Tris, pH 7.4, 2% w/v SDS, 25% v/v glycerol, 10% v/v 2-mercaptoethanol, and 0.01% w/v bromophenol blue), and samples were heated to 105°C for 5 minutes, chilled at 4°C for 10 minutes, and immediately ran on an SDS-polyacrylamide gel. Proteins were transferred to a polyvinylidine difluoride membrane (Bio-Rad) in transfer buffer containing 25 mmol/l Tris, pH 8.8, 192 mmol/l glycine, and 10% v/v methanol. All washing, blocking and antibody solutions were prepared in PBST. Membranes were blocked in 5% milk, followed by overnight incubation with primary antibodies in 1% bovine serum albumin. Membranes were washed three times, followed by secondary antibody incubation for 1 hr in 1% bovine serum albumin. Blots were again washed 3 times, and then probed using an enhanced chemiluminescence system (GE Healthcare) on a GelDoc imager (Bio-Rad). Densitometry was performed following acquisition using ImageJ software (NIH).

Equal amount of proteins (50 μg) from three biological replicates were separated on a 12% SDS-PAGE gel and. The uniformity of the protein amount loaded on the gels was also visulized by staining SDS-PAGE gels using Coomassie Brilliant Blue (Fig. S3). For Western blot analysis, proteins were electrophoretically transferred onto polyvinylidine difluoride membranes (Bio-Rad). The blots were blocked with TBST (20 mM tris-HCl, pH 7.6, 150 mM NaCl, and 0.1%ween-20), containing 5% BSA, followed by incubation with primary antibody solution overnight at 4 °C. After washing with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. Signals were detected with ECL substrate (GE) using Hyperfilm (GE). Supplemental Table S3 represents the primary and secondary antibodies used. Gapdh was used as the loading control. Protein bands were quantified by using ImageJ software. The volume of each band was analyzed using student’s t-test.

Protein preparation and western blotting were conducted as described previously [53 (link)]. Briefly, the MO with TNC was collected and lysed in RIPA buffer containing phenylmethanesulfonyl fluoride and PhosSTOP (Solarbio, China). The supernatant containing proteins was collected and stored at − 80 °C until use. Protein concentrations were measured with a BCA kit (Beyotime, China). Equivalent amounts of the proteins were separated on SDS–PAGE gels and transferred to polyvinylidine difluoride membranes (Bio-Rad, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween 20 (0.5%) for 2 h. Subsequently, the membranes were incubated with the primary antibody at 4 °C overnight and incubated with the secondary antibody at 37 °C for 2 h. The HRP ECL system (Beyotime, China) was used to visualize the protein bands, and the gray values were then analyzed.

Total proteins from VCMs or intact insects were extracted using the sample buffer and separated by 10 or 12% SDS-PAGE, then transferred to polyvinylidine difluoride membranes (Bio-Rad). The membranes were blocked with 5% nonfat milk in PBS with 0.1% Tween 20 and then incubated with RGDV P8-specific IgG, ATG8-specific IgG, ACTB-specific IgG (Purchased from Sigma), or SQSTM1-specific IgG (Purchased from Cell Signal Technology). After incubation with secondary antibody (MultiSciences Biotech), proteins were visualized with the Luminata Classico Western HRP Substrate (Millipore) and imaged with the Molecular Imager ChemiDoc XRS+ System (Bio-Rad).
To confirm RGDV or RDV infection activated the autophagy pathway, autophagy inhibitors (100 nM 3-MA, Sigma; 1 μM BFA, Selleckchem; 20 nM BAF, Enzo) were used to treat the VCMs to inhibit autophagosome formation. VCMs were transfected with (+) and without (-) 3-MA, BFA or BAF for 8 h, and then inoculated with RGDV or RDV at a MOI of 1.0 for 2 h. At 48 hpi, the cells were collected for western blot assay.