Horseradish peroxidase conjugated goat anti mouse igg antibody

For immunoblot analysis, purified NK cells from spleens of naïve mice were lysed using Pierce RIPA buffer (Thermo Fisher Scientific), containing the Complete protease inhibitor cocktail (Roche, Mannheim, Germany). 20 µg of total lysates were separated via non-reducing SDS-PAGE and transferred to PVDF membranes (Carl Roth, Arlesheim, Switzerland) by semi-dry blotting. Membranes were probed with 0.5 µg/ml anti-CD3ζ (6B10.2) at 4°C for 16 h, followed by detection with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Immunoblot signals were generated with Femto-ECL© (Thermo Fisher Scientific).

The preparation of whole cell extract and Western blot is performed as stated [5 (link)]. Protein concentrations were determined by BCA reagent following the manufacturer’s instructions (Bio-Rad, Richmond, CA, USA). All proteins were resolved on sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes. Primary antibodies for AGO2 (no. 2897), phosphor-Akt (Ser473) (no. 9271S), phospho-mTOR (Ser2481) (no. 2974), slug (no. 9585) and vimentin (no. 5741) were purchased from Cell Signaling Technology (Danvers, MA, USA); ABCG2 (no. GTX12131), FAK (no. GTX100764), GAPDH (no. GTX100118) and PTEN (no. GTX101025) were purchased from GeneTex International Co., Ltd. (Hsinchu City 300 Taiwan.); phospho-PI3 kinase p85 (Tyr467/Tyr199)(# bs-3332R, Bioss, Inc., China); mTOR (# 66888-1, Proteintech) and PI3K (P110, #SC-8010) and c-Myc (#SC-40) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish-peroxidase-conjugated goat anti-mouse IgG antibody (Jackson Immunoresearch, West Grove, PA, USA) was used as the second antibody, and the signals were detected using the enhanced chemiluminescence (ECL) system (Millipore, Temecula, CA, USA). Protein band intensities quantified by densitometry (Digital Protein Imagineware, Huntington Station, NY, USA).

Ovaries were quickly dissected from Ae. albopictus adult females at 7, 14, and 21 days postinjection and then homogenized in radioimmunoprecipitation assay lysis buffer. Protein concentrations were measured using a Pierce BCA Protein assay kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. After the addition of 6× SDS loading buffer, the lysates were boiled for 10 min. The proteins of Ae. albopictus ovaries were separated by electrophoresis on a 12% SDS-PAGE gel running at 80 to 130 V for 2 h and then transferred to a polyvinylidene difluoride membrane. After this, the membrane was probed with primary antibodies (1:5,000) and tested using horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (1:10,000, Jackson ImmunoResearch, West Grove, PA). In the experiment, anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) polyclonal rabbit serum (1:10,000, Aksomics, Shanghai, China) was used to monitor equal protein loading. Nitrotetrazolium blue chloride/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) buffer (Sigma-Aldrich) was used to visualize target fragments under room temperature conditions. Anti-ZIKV (SPH2015) envelope antibody (Novus Biological) and anti-Wolbachia hsp60 antibody (Sigma-Aldrich) were used in this study.

Evaluation of oxidative stress, DNA damage responses, and cellular senescence-like state in the cochlear protein extracts from the cochleae of SAMP8 and SAMR1 mice aged 1, 6, and 12 months (n = 16 cochleae per age and per strain) was performed using Western blotting technique as described previously [4 (link)]. Antibodies used included those recognizing Nrf2 (1/1000, Santa Cruz #sc-365949), SOD2 (1/1000, Abcam #ab13533), p66^Shc (1/1000, Abcam #ab33770), phospho-p66^Shc (S36, 1/1000, Abcam #54518), phospho-Chk2 (1/1000, Thr68, Cell Signaling #2661), p53 (1/1500, Cell Signaling #2524), phospho-p53 (1/1500, Ser15, Cell Signaling #9289), p21 (1/2000, Cell Signaling #2946), p16 (1/1000, BD Pharmingen #551154), BubR1 (1/1000, Abcam #ab183496), and p19 (1/1000, Abcam #ab80). β-Actin (1/10,000, Sigma-Aldrich #A1978) served as a loading control. Secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (1/3000, Jackson ImmunoResearch #115-001-003) or goat anti-rabbit IgG antibodies (1/3000, Jackson ImmunoResearch #111-001-003). All experiments were performed in triplicate. Image scans of Western blots were used for semiquantitative analysis.

Ovaries were quickly dissected from Ae. albopictus adult females at 7, 14, and 21 days postinjection and then homogenized in radioimmunoprecipitation assay lysis buffer. Protein concentrations were measured using a Pierce BCA Protein assay kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. After the addition of 6× SDS loading buffer, the lysates were boiled for 10 min. The proteins of Ae. albopictus ovaries were separated by electrophoresis on a 12% SDS-PAGE gel running at 80 to 130 V for 2 h and then transferred to a polyvinylidene difluoride membrane. After this, the membrane was probed with primary antibodies (1:5,000) and tested using horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (1:10,000, Jackson ImmunoResearch, West Grove, PA). In the experiment, anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) polyclonal rabbit serum (1:10,000, Aksomics, Shanghai, China) was used to monitor equal protein loading. Nitrotetrazolium blue chloride/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) buffer (Sigma-Aldrich) was used to visualize target fragments under room temperature conditions. Anti-ZIKV (SPH2015) envelope antibody (Novus Biological) and anti-Wolbachia hsp60 antibody (Sigma-Aldrich) were used in this study.