Anti actin antibody

Proteins were extracted from 30 pooled larvae at 6 dpf. Lysis was performed in ice-cold RIPA buffer [125 mM NaCl, 25 mM Tris-HCl pH 7.4, 1 mM EGTA-Tris pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and complete EDTA-free protease inhibitor cocktail (Roche)]. Crude lysate was cleared by centrifuging for 30 min at 15,000 g and proteins in the supernatant were quantified using a BCA Protein Assay Kit (Pierce). Equal amounts of proteins (50 μg) were loaded on 4-12% Bis-Tris NuPage gels (Life Technologies) and blotted on PVDF Immobilon-P membranes (Millipore). Dried membranes were then washed with TBS buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl) with 1% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) and incubated overnight with the indicated primary antibodies at 4°C: Mitoprofile^® Human WB Total OXPHOS cocktail antibody (1:100; Abcam, ab110411), anti-Grp75 antibody (1:1000; Santa Cruz Biotechnology, H-155), anti-Actin antibody (1:1000; Santa Cruz Biotechnology, AC-15) or anti-Tomm20 antibody (1:10,000; Sigma-Aldrich, HPA011562). Secondary horseradish-peroxidase-conjugated antibodies (Bio-Rad) were incubated for 1 h at room temperature and protein bands were detected by chemiluminescence on a UVITEC Alliance Mini HD9. Quantitation of the signal was performed with ImageJ software (https://imagej.nih.gov/ij/).

Cells and murine lung tissue specimens were homogenized in extraction buffer (50 mM HEPES, 250 mM NaCl, 5 mM EDTA, 0.1% NP-40, 1 mM PMSF,1 mM DTT supplemented with Protease inhibitor cocktail). Whole proteins were extracted by centrifugation (12,000 × g) for 10 min at 4°C. Protein concentration was determined using the BCA kit (Pierce). Equal amounts of protein (20 μg/lane) from the cell lysates were electrophoresed under nonreducing conditions on 10% acrylamide gels. After SDS-PAGE, proteins were transferred to a polyvinylidene difluoride membrane. The membrane was incubated for 2 h in PBS plus 0.1% Tween-20 and 5% nonfat skim milk to block nonspecific binding. Subsequently, the membrane was incubated for 2 h with an antibody against E-cadherin (1:500, Invitrigen), Vimentin (1:500, Sigma) and Twist (1:400, Abcam), followed by incubation with fluorescent secondary antibodies (1:5000, IRDye 800 anti-mouse Molecular Probes, Rockland). Blots were stripped and reprobed by using anti-actin antibody (Santa Cruz Biotechnology). Images were acquired with the Odyssey infrared imaging system and analyzed by the software program as specified by Odyssey.

Cells were lysed in a lysis buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton-X 100 and a Halt^TM Protease Inhibitor Cocktail (100x, Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was measured by a Qubit^TM Protein Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Equal amount of proteins was run on 10% Tris-tricine gel and blotted to polyvinylidene fluoride (PVDF) membrane with a Bio-Rad Wet Blotting System (Bio-Rad Hungary, Budapest, Hungary). The HER2 receptor was detected by an ErbB2 (HER2) antibody cocktail (Thermo Fisher Scientific, MA5-14057, produced in mouse, 1:500) and an anti-mouse-horseradish peroxidase (HRP) secondary antibody (Thermo Fisher Scientific, 32430, produced in goat, 1:500). As a loading control, β-actin was detected by an anti-actin antibody (Santa Cruz Biotechnology, Dallas, TA, USA, sc-1616, produced in goat, 1:2500) and an anti-goat-HRP secondary antibody (Santa Cruz Biotechnology, sc-2354, produced in mouse, 1:2000). After the addition of enhanced chemiluminescence (ECL) substrate (SuperSignal^TM West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific), the chemiluminescent signal was detected by ChemiDoc XRS+ Detection System (Bio-Rad Hungary).