Non fat dry milk

Expression of dynamin-2, caveolin-1 and clathrin after their downregulation by specific siRNA or in A549-shCLTC and A549-shCAV-1 clones was assessed by Western blotting analysis. Cells (3 × 10⁵ cells per sample) were lysed with Laemmli buffer heated to 95°C (150 μL/sample), scraped off the plate, sonicated and boiled (95°C, 3 min). Proteins were separated using 10% acrylamide gel by SDS-PAGE and transferred to nitrocellulose membrane (Amersham Protran, GE Healthcare, USA). The membrane was blocked with 5% (wt/vol) nonfat dry milk (Carl Roth, Germany) in Tris-Buffered Saline containing 1% Tween 20 and probed with the appropriate primary antibodies, followed by incubation appropriate horseradish peroxidase-conjugated IgG secondary antibody. Detection was performed with Pierce ECL Western Blotting Substrate (Thermo Fischer Scientific, USA), using ChemiDoc Imaging System (Bio-Rad, USA). Densitometry was performed with ImageJ software (NIH, USA). The following antibodies were used: anti CLTC (ab21679, Abcam, UK), anti CAV-1 (ab2910, Abcam, UK), anti DNM-2 (ab3457, Abcam, UK). Proteins were normalized to the total protein stained with amidoblack and the results were presented as relative expression of proteins compared to control cells.

Cells were washed twice with PBS and lysed with RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40) containing protease inhibitor cocktail (Roche, Mannheim, Germany) and Phenylmethylsulfonylfluoride (PMSF). Cells were afterwards sonicated (Bandelin SONOPULS) and mixed with Laemmli buffer containing 5% β-mercaptoethanol. After denaturation at 95 °C, they were run through a 15% SDS PAGE mini gel and blotted overnight using a Mini Trans-Blot Electrophoretic Transfer Cell (BioRad, Hercules, CA, USA). Subsequently, the membrane was blocked in 5% non-fat dry milk (Carl Roth, Karlsruhe, Germany) and probed with the primary MPS1 polyclonal antibody (Table S2) followed by a secondary antibody (Table S2). β-actin served as endogenous control (Table S2). Detection was carried out using an electrochemiluminescence solution [53 (link)] and viewed with ImagenFlourChem FC2 (Cell Biosciences, Heidelberg, Australia) with the AlphaView Software (Version 1.3.0.7, ProteinSimple, San Jose, CA, USA). Densitometric analysis was carried out using Image J (NIH, Bethesda, MD, USA).

Fifteen micrograms of cell lysate protein was denatured in 1x SDS-loading dye at 95°C, separated by SDS-PAGE and transferred onto a PVDF membrane (pore size: 0.45 μm; Carl Roth, Karlsruhe, Germany) according to standard protocols. Membranes were blocked with 10% nonfat dry milk (Carl Roth) in 1xTST buffer (50 mM Tris HCl, 0.1% Tween 20, 150 mM NaCl; pH 7.4) and then incubated with anti-Flag epitope peroxidase-conjugated antibody (1:8,000; A8592, Sigma-Aldrich) or anti-His (1:3,000; 18184, Abcam). GAPDH (1:20,000; 2118S, Cell Signaling, Danvers, MA) was used as a loading control. Immunoblots were visualized using the Clarity Western ECL plus Western Blotting Detection Reagent (Fisher Scientific) and ChemiDoc Touch Imaging System (Bio-Rad).

Protein isolation and Western blotting were performed as previously described [20 (link)]. KGN cells were lysed using RIPA buffer plus protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, USA). A total of 7 µg (NOX4) or 20 µg (cleaved caspase 3, clCASP3) protein per lane was loaded on a 10% (NOX4) or 12% (clCASP3) SDS gel and run (NOX4: 20 min at 100 V + 70 min at 120 V; clCASP3: 20 min at 75 V + 40 min at 150 V). After blotting (NOX4: 55 min at 100 V; clCASP3: 60 min at 100 V) and blocking with 5% non-fat dry milk (Roth, Karlsruhe, Germany) in Tris-buffered saline with Tween 20 (5 mM Tris, 100 mM NaCl, 0.05% Tween 20, pH 7.5), rabbit anti-NOX4 polyclonal antiserum (1:1000, #7927, ProSci, Fort Collins, CO, USA) or rabbit anti-clCASP3 monoclonal antibody (1:1000, #9664, Cell Signaling Technology, Danvers, MA, USA) were administered to detect these proteins. As a loading control, mouse anti-β-actin monoclonal antibody (1:10000, #A5441, Sigma-Aldrich) was used. HRP-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (Jackson ImmunoResearch Europe, Cambridgeshire, UK) were used to visualize specific binding. Band intensities were determined using the FIJI software [21 (link)].

Confluent cells were washed once with ice-cold PBS and lysed with 1 × PhosphoSafe lysis buffer (Merck Millipore). Cell lysates were sonicated and centrifuged for 10 min at 14,000× g and 4 °C following determination of total protein concentration in supernatants using BioRad DC protein assay. Samples containing equal amounts of protein were prepared using 3× SDS sample buffer and 125 mM dithiothreitol (both from New England Biolabs, Ipswich, MA, USA), 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 nonfat dry milk (Carl Roth GmbH, Karlsruhe, Germany) or BSA (Sigma-Aldrich) and incubated with primary antibodies at 4 °C overnight. HRP-linked secondary antibodies and Amersham ECL Prime Detection Reagent (GE Healthcare Dharmacon) were used for detection of proteins on a BioRad ChemiDoc XRS imaging system. RotiFree stripping buffer (Carl Roth GmbH) was used for membrane stripping.

Confluent cells were washed once with ice-cold PBS and lysed with 1x PhosphoSafe lysis buffer (Merck Millipore). Cell lysates were sonicated and centrifuged for 10 min at 14.000 × g and 4 °C following determination of total protein concentration in supernatants using BioRad DC Protein Assay. Samples containing equal amounts of protein were prepared using 3x SDS Sample Buffer and 125 mM Dithiothreitol (both from New England Biolabs), 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 nonfat dry milk (Carl Roth GmbH, Karlsruhe, Germany) or BSA (Sigma-Aldrich) and incubated with primary antibodies at 4 °C overnight. HRP-linked secondary antibodies and Amersham ECL Prime Detection Reagent (GE Healthcare) were used for detection of proteins on a BioRad ChemiDoc XRS imaging system. RotiFree stripping buffer (Carl Roth GmbH) was used for membrane stripping. Signal intensities were quantified by densitometry and computed with either NIH image J or Image Lab (version 5.2.1, BioRad).