Mini protean tgx any kd precast gels

We performed two dimensional (2D) SDS-PAGE gel electrophoresis experiments using each of the four A. contortrix venoms to compare venom compositional profiles. For each gel, 0.5 mg of venom was prepared for 2D gel electrophoresis using the ReadyPrep™ 2-D Cleanup Kit for isoelectric focusing (IEF) (Bio-Rad) as per the manufacturer’s instructions. Cleaned-up venom samples were then applied to 7 cm, pH 3–10, non-linear IPG strips (Bio-Rad) using the ReadyPrep™ 2-D starter kit (BioRad), as per manufacturer’s instructions, and re-hydrated overnight at room temperature. After re-hydration, IEF was performed using a PROTEAN^® IEF Cell (Bio-Rad) with the manufacturer’s standard electrophoresis protocol for 7 cm IPG strips (default cell temperature = 20 °C; maximum current 50 Ua/strip; voltage = 250 V with linear ramp for 20 min; 4000 V with linear ramp for 2 hours; 4000 V with rapid ramp for 10,000 V-hr). After IEF, IPG strips were equilibrated (as per the ReadyPrep™ 2-D starter kit) and loaded onto Mini-PROTEAN TGX AnyKd precast gels (Bio-Rad) and run at 200 V for 35 minutes. Gels were then rinsed in water and stained with G-250 coomassie blue stain (Bio-Rad) for 1 hr to visualise proteins. Original (unedited) gel images can be found in Fig. S2.

Cells were harvested and lysed in Nonidet P-40 buffer containing a protease-inhibitor mixture (Sigma). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on Mini-PROTEAN TGX Any kD Precast Gels (BioRad, Hercules, CA, USA) and subsequently transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Immunoblots from tumor cell lysates were probed with antibodies against DDX3X (Sigma), EGFR, phospho-EGFR (Tyr1068), phospho-EGFR (Tyr1173), phospho-EGFR (Tyr845), Akt, phospho-Akt, Erk1/2, phospho-Erk1/2, and β-actin (Sigma). All antibodies except for anti-DDX3X and anti-β-actin were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Secondary antibodies consisted of anti-mouse IgG (BioRad) and anti-rabbit IgG conjugated to horseradish peroxidase (Abcam, Cambridge, MA, USA). Immunoreactive protein bands were visualized using an ECL kit (Pierce). At least three independent experiments were performed for all analyses.

Cell lysis and immunoblotting was essentially performed as described previously [8 (link)]. In brief, cells were washed once with ice-cold PBS and lysed with 1× PhosphoSafe lysis buffer (Merck Millipore). Following clearance of the lysates by centrifugation, their total protein concentrations were determined with the DC Protein Assay (BioRad). Equal amounts of proteins were fractionated by polyacrylamide gel electrophoresis on mini-PROTEAN TGX any-kD precast gels (BioRad) and blotted to PVDF membranes. Membranes were blocked with nonfat dry milk or BSA and incubated with primary antibodies either for 2 h at RT or overnight at 4 °C. After washing and incubation with HRP-linked secondary antibodies, chemoluminescent detection of proteins occurred on a ChemiDoc XRS imaging system (BioRad) with Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany).

The immunoblotting procedure was described in detail in earlier publications [15 (link),16 (link),17 (link),18 (link)]. An amount of 20–40 μg of total cellular protein quantified with the DC Protein-Assay Kit (BioRad) was fractionated by SDS-PAGE on mini-PROTEAN TGX any-kD precast gels (BioRad) and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Eschborn, Germany) equilibrated with methanol and transferred to blotting buffer. After blotting, membranes were blocked with Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% bovine serum albumin. Following overnight incubation with the primary antibody at 4 °C in TBST, the primary antibody was removed by washing the blots with TBST. Subsequently, blots were incubated with the appropriate peroxidase-conjugated secondary antibodies and developed with the chemiluminescent detection kit (Amersham ECL Prime Detection Reagent, Cytiva, Marlborough, MA, USA) following the manufacturer’s protocol on a ChemiDoc XRS imaging system (BioRad). Signal quantifications for the proteins of interest and HSP90 were done by densitometry using either the built-in function of the ChemiDoc XRS system or the program Image Lab 5.2.1. The antibodies used are listed in Section 2.1.

Cell lysis and immoblotting was essentially performed as described previously [18 (link)]. In brief, cells were washed once with ice-cold PBS and lysed with 1× PhosphoSafe lysis buffer (Merck Millipore). Following clearance of the lysates by centrifugation, their total protein concentrations were determined with the DC Protein Assay (BioRad). Equal amounts of proteins were fractionated by polyacrylamide gel electrophoresis on mini-PROTEAN TGX any-kD precast gels (BioRad) and blotted to PVDF membranes. Membranes were blocked with nonfat dry milk or bovine serum albumin and incubated with primary antibodies either for 2 h at RT or overnight at 4 °C. After washing and incubation with HRP-linked secondary antibodies, chemoluminescent detection of proteins was done on a ChemiDoc XRS imaging system (BioRad) with Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The signals for the proteins of interest were normalized to those for GAPDH or HSP90 (identity verified by size determination using the SM1841 molecular weight marker from Fermentas/Thermo Fisher Scientific (Supplementary Figure S7)).

Protein expression levels were determined by western blot analysis. Briefly, CT26, A549, S2, H1975, HCC827 and Lu85 cells were lysed with 10% sodium dodecyl sulfate. Mouse kidney, human kidney and HK-2 cell lysates were used as positive controls for western blotting. The cell lysates were centrifuged, and the supernatants were collected to measure the amount of protein using BCA protein assay reagent (Thermo Scientific, Waltham, MA, USA). Equal amounts of total protein from each sample were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on Mini-PROTEAN TGX Any kD Precast Gels (Bio-Rad, Hercules, CA, USA), after which the proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and immunostained with primary antibodies against megalin^{24 (link)} and mouse β-actin (AC-15, Abcam, England). The secondary antibody was horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Bio-Rad, CA, USA). HRP was detected using the ECL western detection kit (Pierce, Rockford, IL, USA).

Confluent cells were washed once with ice-cold PBS and lysed with 1 x PhosphoSafe lysis buffer (Merck Millipore). Following sonication and clearing, the total protein concentration of the supernatants was determined with the BioRad DC Protein Assay. Samples were subjected to gel electrophoresis using BioRad mini-PROTEAN TGX any-kD precast gels and blotted to 0.45 μm PVDF membranes. Membranes were blocked with non-fat dry milk or BSA and incubated with primary antibodies at 4 °C overnight. HRP-linked secondary antibodies and Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany) were used for chemoluminescent detection of proteins on a BioRad ChemiDoc XRS imaging system.