Trans blot turbo midi pvdf transfer pack

Meningioma cell lines were harvested and lysed with RIPA buffer (Thermo Scientific, USA) and halt Protease Inhibitor Cocktail (Thermo Scientific, USA). Equal amounts of denatured protein sample were separated by SDS-PAGE and were then transferred to PVDF membranes using Trans-Blot® Turbo™ Midi PVDF Transfer Packs (Bio-Rad, USA) for immunoblot analysis. We used antibodies to recognize PD-L1 (1:200 dilution, Cat# ab58810, Abcam, USA) [47 , 48 (link)], GAPDH (1:500 dilution, Cat# sc-32233, Santa Cruz, USA) as loading control. All protein bands were detected using Western Blotting Luminol Reagent (Cat# sc-2048, Santa Cruz, USA).

Total protein was extracted from microglia by scraping the cells in SDS/sample buffer. Tissue from cerebellar organotypic slices was directly heated at 100°C in sample buffer (7 min). Samples were loaded and size‐separated by electrophoresis using Criterion TGX Precast 12% gels and transferred to Trans‐Blot Turbo Midi PVDF Transfer Packs (Bio‐Rad, Hercules, USA). Membranes were blocked in 5% skimmed milk and 5% serum in Tris‐buffered saline/0.05% Tween‐20 (TBS‐T) and proteins detected by specific primary antibodies to BDNF (#sc‐547, 1:200; Santa Cruz), to MBP (#SMI‐99P, 1:2,000, Covance), to GAPDH (#MAB374, 1:2,000; Millipore), and to β‐actin (#A2066, 1:1,000; Sigma), followed by secondary peroxidase‐coupled goat anti‐rabbit antibodies (#A6154, 1:2,000; Sigma) or sheep anti‐mouse antibodies (#A6782, 1:2,000; Sigma). After washing, blots were developed using an enhanced chemiluminescence detection kit according to the manufacturer's instructions (SuperSignal West Dura or Femto, Pierce). Images were acquired with a ChemiDoc MP system (Bio‐Rad) and quantified using ImageJ software. Values of BDNF and MBP were normalized to corresponding β‐actin and GAPDH signal, respectively.

Aliquots of approximately 50 mg of tissue were mixed with 1 ml of lysis RIPA buffer (R 0278, Sigma, Saint Louis, MO) buffer containing a protease inhibitor cocktail (11 836 170 001, Roche Diagnostics GmbH, Mannheim, Germany), 8 mM sodium orthovanadate, and 0.2 mM phenylmethanesulfonyl fluoride. Protein extracts were collected after tissues were homogenized (1 min) and centrifuged (20000 × g, 20 min). Aliquots of 30 µg of protein were mixed with an equal volume of 2 X SDS-PAGE sample loading buffer, and resolved by SDS-PAGE. Resolved samples were transferred to polyvinylidene fluoride (PVDF) membranes by using Trans-Blot^® Turbo™ Midi PVDF Transfer Packs, 170-4157 BioRad, Hercules, CA). Membranes were blocked with 5% nonfat dry milk in Tris-Buffered Saline with Tween 20 (1X TBST). Blots were incubated overnight at 4°C with primary antibodies (EGFR Tyr 1068, ERK, pERK1/2 Thr202/Tyr204, AKT, pAKT Ser 473, pS6 Ser235/236, and β-actin) at 1:1000 dilutions, followed by incubation with a 1: 4000 dilution of HRP-linked anti-rabbit IgG secondary antibodies for one hour . All antibodies used for blotting were from Cell Signaling Technology, Danvers, MA. Immunoreactive protein bands were detected by ECL-Prime Western blotting detection reagent (RPN2236, GE Healthcare, Little Chalfont, UK).

Antibodies were purchased as follows: IGF-II Receptor/CI-M6PR (D3V8C) and β-actin (# 8H10D10) (Cell Signaling Technology, Danvers, MA, USA), α-Gal-A (#GTX101178) (GeneTex, Irvine, CA, USA). Whole-cell extracts (WCEs) were prepared in radioimmunoprecipitation (RIPA) buffer. Protein concentrations were determined using the BCA Protein Assay Kit (ThermoFisher, Rockford, IL, USA). Thirty micrograms (30 μg) of WCE were separated on mini protein TGX stain-free gel (Bio-Rad, Hercules, CA, USA) and electroblotted using the Trans-Blot^® Turbo™ Midi PVDF Transfer Packs (Bio-Rad, Hercules, CA, USA). Membranes were diluted with antibodies (1:1000 dilutions) in 5% BSA, 1 × TBS, 0.1% Tween20, and gently shaking overnight at +4 °C. The ChemiDocTM MP Imaging system (Bio-Rad) was used to visualize and quantitate optical density (IOD). The IODs of bands of interest were normalized to the loading control actin used in the same blot [8 (link)], and the normalized value of the controls was set to 1 for a comparison between separate experiments.

To confirm the presence of T. cruzi-specific proteins in EV secreted by trypomastigotes, Western blots using anti-trans-sialidase (mAb39) antibody were performed, following the methodology described by Retana Moreira et al. in 2021^{13 (link)}. For the analysis, 30 µg of EV of trypomastigotes were resolved by SDS-PAGE, transferred to PVDF membranes (Trans-Blot Turbo Midi PVDF Transfer Packs, Bio-Rad Laboratories, USA) for 40 min at 40 V in a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, USA), and blocked overnight with 5% non-fat milk in PBS-0.1% Tween 20. The membranes were then washed in PBS-0.1% Tween 20 and incubated overnight at 4 °C with the primary antibody anti-TS mAb 39 (1:1000) (produced in mice). After the incubation, the membranes were washed in PBS-0.1% Tween 20 and incubated for 1 h with secondary antibody goat anti-mouse IgGs conjugated with peroxidase (1:1000) (Agilent Technologies, Santa Clara, CA, USA). The reaction was visualized using Clarity ECL Western substrate (BioRad, Hercules, CA, USA) in a ChemiDoc Imaging System (BioRad, Hercules, CA, USA).