Immun blot pvdf

Protein concentration of the cell wall protein extracts from 1 g of hypocotyl tissue was estimated with Qubit Protein Assay Kit using a Qubit 3 Fluorometer (Thermo Fisher Scientific). Extracellular samples containing 10 μg of protein were incubated with 5 × SDS sample buffer for 10 min at 95°C, resolved by SDS-PAGE under reducing conditions and electroblotted to 0.2 μm PVDF membranes (Immun-Blot PVDF, Bio-Rad). Blots were blocked with 5% (w/v) skim milk powder in Tris-buffered saline (TBS; 150 mm NaCl, 10 mm Tris, 0.1% (v/v) Triton X-100, pH 7.6) and incubated for 2 h with a 1:1000 diluted polyclonal rabbit anti-PME18 antibody conjugated to horseradish peroxidase (Bioss Antibodies, United States). Detection was performed using a colorimetric assay with 3,3′-diaminobenzidine tetrahydrochloride (Roth) as a substrate. Western blotting was performed for two independent biological replicates with 90 plants per combination, each with three technical replicates. Blots were scanned using a flatbed scanner (Hewlett-Packard Color LaserJet Pro M177), and protein quantity was estimated with ImageJ software (Schneider et al., 2012 (link)) by measuring pixel intensity (mean gray value) of the detected signal. Protein loading was visualized by membrane staining with 0.5% (w/v) Ponceau S in 1% acetic acid.

Immunoprecipitates were made using antibodies to MCL-1 (ThermoScience MA5-13932), BCL-2 (Millipore 05-729) and BCL-XL (CST 2764) using the TrueBlot kit according to the manufacturer’s instructions (Rockland Immunochemicals). Then 20 μg reduced protein was loaded in each well of 12% NuPAGE SDS polyacrylamide gels (Life Technologies) and separated using electrophoresis. Proteins were transferred to Immun-Blot PVDF (Biorad) and analyzed by western blot for mouse MCL-1 (CST 5453), BCL-2 (Millipore 05-729), BIM (CST 2933), PUMA (CST 4976), NOXA (ENZO ALX-804-C10), p53 (Epitomics 1026-1), Cofilin (total) (CST 5175), S3-Cofilin (CST 3313) and beta-ACTIN (Santa Cruz AC-74, A5316).

Cell lysates (10–20 μg of protein) were fractionated by polyacrylamide gel electrophoresis 10–12% (SDS-PAGE) denaturant, and separated according to different molecular weights (Standard Precision Plus Protein Kaleidoscopic™, Bio-Rad, Hercules, CA, USA). The proteins were transferred (25 V, 1.0 A, and 30 min) using a transfer buffer with methanol and polyvinylidene difluoride membrane (Immun-Blot^® PVDF, Bio-Rad) on Trans-Blot Turbo™ equipment (Bio-Rad). Then, the membranes were washed with 5% BSA (Sigma-Aldrich, St. Louis, MI, USA) in 0.1% T-TBS and incubated overnight at 4 °C in the presence of the following primary antibodies: anti-arginase (gt5811-rabbit-1:1000) (Thermo Fisher Scientific, Waltham, MA, USA), anti-caspase-3 (sc7148-rabbit-1: 250) (Santa Cruz Biotechnology, Dallas, TX, USA), or anti-β-actin (a5441-mouse-1:5000) (Sigma-Aldrich). The membranes were incubated for at least one hour with a specific peroxidase-conjugated anti-rabbit secondary antibody (ab6789-mouse or ab6721-rabbit-1:10,000) (Abcam, Cambridge, MA, USA). Immunoreactive proteins were visualized by detecting their chemiluminescence through ECL (GE) solution. Membranes were developed and photographed using ImageQuant™ LAS500 (GE Healthcare, Buckinghamshire, England), and densitometry was quantified using ImageJ software.

Samples were loaded on a 10% SDS-gel for electrophoresis and transferred onto a membrane (Immun-Blot PVDF, Biorad). Western-blot analysis was performed according to standard protocols using the following antibodies: anti myc-tag (1:200, Santa Cruz Biotechnology, Dallas, TX, Cat# sc-47694 RRID:AB_627266), anti-α-tubulin (1:50.000, DM1A, Sigma-Aldrich Cat# T9026 RRID:AB_477593) and anti-Barren (1:3000, kindly provided by Hugo Bellen, (Bhat et al., 1996 (link)), RRID:AB_2567044).

Yeast strains were analyzed by Western blotting according to standard protocols [109] (link). For Western analysis, 10 µL of protein sample were separated via SDS-PAGE and transferred to Immun-Blot PVDF (Bio-Rad) using standard methods. Protein detection was carried out using antibodies against Hemagglutinin (HA) (1∶2000; Abcam) in TBS+0.1% Tween20 and 5% milk. After immunodetection of Hemagglutinin, the membrane was stripped using Stripping Buffer (62.5 mM Tris pH 6.8, 100 mM β-mercaptoethanol, and 2% SDS) at 65°C for 30 minutes with occasional agitation. Normalization of loading was achieved by probing the original membrane with antibodies against yeast 3-phosphoglycerate kinase Pgk1p (1∶5000; Invitrogen) in the buffer conditions used previously.