Hrp conjugated anti rabbit igg

Cell pellets were resuspended in PBS and lysed in 2× Laemmli sample buffer (Sigma-Aldrich, catalog S3401). Proteins were resolved by electrophoresis in a 4%–12% Bis-Tris Plus gel (Life Technologies, catalog NW04120) and transferred to a polyvinylidene difluoride (PVDF) membrane overnight. Membranes were blocked with 5% nonfat milk in TBST (Tris-buffered saline + 0.1% Tween 20). Membranes were incubated with primary antibody for 2 hours at room temperature, washed with TBST, and then incubated for 1 hour at room temperature with the secondary antibody, horseradish peroxidase–conjugated (HRP-conjugated) anti-rabbit IgG (Invitrogen, catalog 31460; 1:10,000 dilution). Immunoreactive material was visualized using Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, catalog 32132) and imaged with a ChemiDoc Touch Imaging System (Bio-Rad). Quantitation was performed using Image Lab Software (Bio-Rad) by normalizing each RPS19 band to an actin loading control.

Primary antibodies used in the study include the following: mouse monoclonal anti-β-actin (Cell Signaling), mouse monoclonal anti J2 double stranded RNA (dsRNA; English and Scientific Consulting Kft, Szirák, Hungary), rabbit polyclonal anti-SARS-CoV-2 spike (Sino Biological, #40591-V08H) and rabbit polyclonal anti-MERS spike (Sino Biological, #40069-RP02). The secondary antibodies used for immunofluorescence were Alexa Fluor 488 goat anti-mouse. The secondary antibodies used for western blot analyses were HRP-conjugated anti-rabbit IgG (Invitrogen, Waltham, MA, USA) and HRP-conjugated anti-human IgG (Invitrogen).

To examine the expression of CD44 in the two human ovarian cancer cell lines [A2780 and SKOV3], total cell protein extract was isolated from 2 × 10⁷ cells in a lysis buffer containing 50 mM Tris at pH = 7.5, 50 mM β-mercaptoethanol, 0.5% Triton ×100, and 1 mM EGTA in DDW. Following 1 hr incubation on ice, the extract was centrifuged at 14,000xg for 30 min at 4°C and the supernatant was collected. Protein content was determined using the Bio-Rad protein assay. Protein aliquots (60 μg) were resolved by electrophoresis on 10% polyacrylamide gels containing SDS and electroblotted onto a Protran BA83-cellulose nitrate membrane (Whatman). The blots were then blocked for 1 hr at room temperature in Tris buffered saline (TBS) containing 150 mM NaCl, 20 mM Tris-base at pH 7.6 containing 1% skim milk. For the detection of CD44, rabbit monoclonal antibody (EPR1013Y) was used at a dilution of 1:5,000. Blots were then rinsed and reacted with a secondary antibody consisting of horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:10,000 dilution, Invitrogen, Carlsbad, CA) for 1 hr at room temperature. Following three 10-min washes in TBS at room temperature, enhanced chemiluminescence detection was performed. To normalize for any differences in actual protein loading, the blots were stripped and re-probed with a monoclonal antibody to β-actin (1:4,000 dilution; Chemicon, Temecula, CA).

Proteins were separated by electrophoresis on 12 % Tris-glycine polyacrylamide gel at 150 V for 1 h 30 min, (for Iba-1 detection) and on 4–12 % polyacrylamide precast NuPAGE Bis-Tris gel (Invitrogen) at 200 V for 2 h 30 min (for GFAP detection), and transferred onto a PVDF membrane (Bio-Rad, Hercules, CA) using a semi-dry blot system (Bio-Rad) at 25 V for 50 min. Membranes were blocked in PBS/2 % fat-free dry milk overnight at 4 °C and then incubated with primary antibodies (rabbit anti-β- actin and rabbit anti-GFAP [1:1000] or rabbit anti-Iba-1 [1:2000]) diluted in PBS/milk/0.2 % Triton X-100 overnight at 4 °C. After washing with PBS/0.2 % Tween, membranes were incubated with HRP-conjugated anti-rabbit IgG (Invitrogen) for 2 h at room temperature. Immunoreactive bands were detected by chemiluminescence using the Super Signal West Dura Extended Duration Substrate kit (Pierce, IL). The NIH ImageJ software was used for densitometric analysis of bands. β-actin served as an internal control.

Western blotting was performed using a standard protocol. We used the following primary and secondary antibodies: PCNA (1:1000; ab92552, Abcam, Cambridge, MA, USA), CYCLIN D1 (1:1000; #55506, Cell Signaling Technology, Danvers, MA, USA), phospho-CDK2 (p-CDK2; 1:1000; #2561, Cell Signaling Technology, Danvers, MA, USA), CDK2 (1:1000; #18048, Cell Signaling Technology, Danvers, MA, USA), SIRT1 (1:1000; #9475, Cell Signaling Technology, Danvers, MA, USA), PGC1α (1:1000; NAP1-04676, Novus Biologicals, Littleton, CO, USA), NRF1 (1:1000; #46743, Cell Signaling Technology), mtTFA (1:1000; ab131607, Abcam, Cambridge, MA, USA), ACTIN (1:2000; sc-8432, Santa Cruz Biotechnology, Dallas, TX, USA), ACTB (1:2000; A5316), HRP-conjugated anti-rabbit IgG (1:5000; #65-6120, Invitrogen, Carlsbad, CA, USA) and anti-IgG (1:5000; #62-6520, Invitrogen, Carlsbad, CA, USA). Bands were detected using a ChemiDoc XRD+ system (Bio-Rad, Hercules, CA, USA), and relative intensity was assessed using Image Lab software (Bio-Rad, Hercules, CA, USA).

Cell lysates generated from treated moDCs as above were precipitated by addition of 4x volume of acetone (Sigma-Aldrich) and incubation at −20 °C for 24 h. Samples were centrifuged for 15 min at 12,000g at 4 °C. Pellets were resuspended in LDS sample buffer (Invitrogen) at a concentration of 2.5 mg/mL. 25 µg of each lysate was analyzed by SDS-PAGE on 10% Bis-Tris gel (Invitrogen) or 12.5% Bis-Tris gel containing 50 μmol/L SuperSep Phos-tag™ reagent (Wako Chemicals) Separated proteins were transferred to PVDF membrane (Invitrogen) and incubated with primary antibodies as indicated; anti-Sec22b (Novus Biologicals), anti-PKC delta (Abcam), anti-PKC delta phospho-S645 (Abcam), anti-STAT1 (Abcam), anti-STAT1 phospho-S727 (Abcam), anti-GAPDH (Abcam), anti-STX4 (BD Biosciences), anti-PSME2 (Invitrogen), anti-NCF2 (Invitrogen) for 18 h at 4 °C. Membranes were washed and incubated with HRP-conjugated anti-rabbit IgG (Invitrogen) or anti-mouse IgG (Cell Signaling Technology) at room temperature for 1 h. Chemiluminescent substrate (SuperSignal™ West Pico PLUS; Thermo Fisher) was used for photodetection of target proteins.