0.22 μm nitrocellulose membrane

The expression of surface streptavidin was first detected by western blotting. Secreted streptavidin proteins in the Lp culture were precipitated with 15% trichloroacetic acid (TCA), separated via SDS-PAGE, and transferred onto a 0.22μm nitrocellulose membrane (Bio-Rad). The transferred proteins were visualized on the western blot through detection by a primary c-Myc antibody at a 1:1000 dilution in 3% BSA and a secondary anti-mouse IgG (Cell Signaling #7076) at a 1:1000 dilution in 3% BSA. The membrane-bound streptavidin proteins were dissolved in 8 M urea and visualized using an anti-HA antibody (Thermo Fisher 26183) at a 1:1000 dilution in 3% BSA and the same secondary anti-mouse IgG antibody.
After the confirmation of protein expression, engineered Lp strains were incubated with c-Myc monoclonal or HA tag antibody (1:500 dilution in 1% BSA) for one hour, followed by a one-hour incubation with the secondary anti-mouse antibody (1:500 dilution in 1% BSA) at 25 °C. The antibody-treated cells were then visualized by a Leica DM4000 B fluorescence microscope or analyzed in the CytoFLEX analyzer.

Cells were harvested and washed with PBS, then lysed with RIPA buffer (Millipore) containing Halt Protease/Phosphatase Inhibitor Cocktail and 5 mmol/L ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific). Lysate was spun at 14,000 rpm for 15 minutes to pellet cell debris, and protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein lysates were diluted with RIPA buffer and 4x Laemmli buffer prepared with β-mercaptoethanol (Bio-Rad) to a final concentration of 3 μg protein/μL and heated to 95°C for 5 minutes in a heat block. Protein samples were run in 12% SDS-PAGE gels, followed by transfer onto 0.22 μm nitrocellulose membrane (Bio-Rad) using Towbin buffer with 20% methanol. Membranes were blocked using 5% milk diluted in Tris-buffered saline with 0.1% Tween 20 (TBST). Primary antibodies (Cell Signaling Technology) were generally diluted 1:1,000 in 5% BSA Fraction V (Thermo Fisher Scientific) in TBST as recommended by the manufacturer; GAPDH and β-actin antibody were purchased from Santa Cruz Biotechnology and diluted 1:10,000 in 5% BSA in TBST. Secondary antibodies (Cell Signaling Technology) were diluted in 5% milk in TBST. Membranes were imaged using either a Konica Minolta SRX-101A developer or a ChemiDoc XRS+ System imager (Bio-Rad).

Cell pellets were collected 24 h after treatment, protein samples were fractionated on 4–12% Criterion™ XT Bis-Tris gels (Bio-Rad, 3450124) in 1X-XT MOPS buffer (BioRad, 161-0788), and transferred to 0.22 μm nitrocellulose membranes (Bio-Rad, 1620112). Antibodies used in this study were directed against: GLUT1 (Fisher, PA5-16793), GLUT4 [IF8] (Santa Cruz, sc-53566), GS (BD Biosciences, 610517), GLS1 [EP7212] (Abcam, ab156876)—to assess for GAC and KGA isoforms, β-Actin (Santa Cruz, sc-1616). Western Blot band quantification was performed using ImageJ 1.49v software (NIH, USA).

Samples of PRRSV‐infected and DMEM‐inoculated PAMs were lysed at 24 h post infection and protein concentrations were determined. Samples (20 μg) were separated by 12% SDS‐PAGE and transferred to 0.22 μm nitrocellulose membranes (Bio‐Rad Laboratories, Hercules, CA, USA). Membranes were blocked with 5% skim milk in Tris‐buffered saline containing 0.05% Tween‐20 and incubated overnight at 4 °C with monoclonal antibodies against heat shock protein 70 (HSP70; ab5439; Abcam plc, Cambridge, UK) or KDEL receptor (ab69659; Abcam plc). After washing three times, membranes were incubated at 37 °C for 60 min with horseradish peroxidase‐conjugated anti‐mouse IgG or anti‐rabbit IgG (Abcam plc). Detection used chemiluminescence luminal reagents (Pierce Biotechnology, Waltham, MA, USA).

Cells and tissues were lysed with radio immunoprecipitation assay (RIPA) buffer, mixed with Laemmli sample buffer (1×), and boiled. Proteins were subjected to 10% SDS-PAGE and electroblotted onto 0.22-μM nitrocellulose membranes (BioRad Laboratories, USA). Membranes were blocked with Tris-buffered saline plus 0.2% Tween 20 (TBS-T) containing 3% BSA (Sigma Aldrich, USA), followed by overnight incubation with the primary antibody and washing with TBST buffer. Then, membranes were incubated with the secondary antibody (anti-mouse, HRP conjugate, 1:10000 Sigma Aldrich, USA), diluted in blocking buffer for 1 h at room temperature, and washed again with TBS-T. Antibody-reactive proteins were detected with enhanced chemiluminescence, Amersham ECL Plus western blotting detection reagents (GE health care, UK). RUNX2, CDK4, MMP2, and GAPDH antibodies were purchased from Santa Cruz Biotechnology (USA).

Whole mouse brains were quickly frozen on dry ice and stored at − 80 °C before extraction. Tissue from eight mice were thawed, individually sonicated in 4% SDS/50 mM Tris, pH 7.6, and heated for 10 min at 90 °C. Protein concentrations for all fractions were determined using the BCA assay (Pierce, Waltham, MA, USA), using bovine serum albumin as a standard. Samples were normalized for total protein content and SDS-containing sample buffer was added to samples, which were then further heated for 10 min at 90 °C. Protein samples were separated on SDS-polyacrylamide gels (8% or 15%) and transferred electrophoretically onto 0.22 μm nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were blocked in 5% non-fat milk in Tris-buffered saline, pH 7.6 (TBS) for 1 h at room temperature, then incubated in primary antibodies (detailed in Table 2) diluted in 5% non-fat milk/TBS block solution overnight in 4 °C. After incubation, membranes were rinsed with agitation in TBS for 5 min, repeated eight times. Membranes were then incubated with goat anti-mouse secondary antibody conjugated to horseradish peroxidase (Jackson Immuno Research Labs, Westgrove, PA), diluted 1:1000 in 5% non-fat milk/TBS for 2 h at room temperature. Protein band signal was detected with Western Lightning-Plus ECL reagents (PerkinElmer, Waltham, MA) and chemiluminescence imaging (PXi, Syngene, Frederick, MD).