Ecl advanced

Western blotting was performed as described.¹⁴ Cultured cells (1 × 10⁶) were lysed in 500 µL lysis buffer (20 mM Tris‐HCl, 150 mM NaCl, 1% Triton X‐100, 1 mM EDTA, 1 mM Na2VO4, 1 mM PMSF, 10 mM 1,10‐phenanthroline monohydrate) supplemented with 1‐fold Complete Inhibitor (Roche), incubated for 10 minutes and cleared by centrifugation at 16 000 g for 5 minutes. Cell lysates containing 15‐20 µg protein (according to bicinchoninic acid assay, Thermo Fisher/Pierce) were heated in SDS reducing sample buffer (250 mM Tris‐HCl (pH 6.8), 50% (w/v) glycerol, 10% (w/v) SDS, 0.1% bromophenol blue and 5% β‐mercaptoethanol) and subjected to SDS‐polyacrylamide gel electrophoresis using 10% Tris‐glycine gels. Proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes (Hybond‐P, Amersham; 10 600 022). Membranes were blocked with 5% (w/v) non‐fat dry milk in Tris‐buffered saline with 0.05% Tween. For analysis of phosphoproteins, dry milk was substituted by 5% bovine serum albumin (BSA). Membranes were probed overnight with primary antibodies followed by incubation with POD‐coupled secondary antibodies (diluted 1:20 000). After addition of chemiluminescence substrate (ECL advanced, Amersham), signals were recorded using luminescent image analyser LAS2000 (Fujifilm, Tokyo, Japan) and quantified using open source ImageJ software (Wayne Rasband, NIH).

Kidney tissue was homogenized in lysis buffer (50 mM HEPES, 250 mM NaCl, 5 mM EDTA, 0,1% Nonidet P-40) using polytron homogenizer. After centrifugation for 10 min at 10000 rpm, 4°C, supernatant was collected and protein concentration was determined using a BCA protein assay kit (Bio-Rad). 25μg of proteins were electrophoresed on 12% SDS-PAGE gels and transferred to PVDF membranes (Immobilon-P, Millipore), as previously described.[20 (link)] Membranes were probed with primary antibodies against CalbindinD28k (1:10000) and α-tubulin (1:5000) over night at 4°C. Appropriate horseradish peroxidase-conjugated secondary antibodies were used at 1:10000. The immunoreaction was visualized using chemiluminescent kits EZ ECL (Biological Industries) or ECL Advanced (Amersham Biosciences). Images were digitally acquired by Chemidoc (Bio Rad). Positive immunoreactive bands were quantified by densitometry and compared with the expression of adequate loading control (α-tubulin).

For western blot, 25–50 oocytes per sample were collected. Alternatively, cell lysates (input) or protein immunoprecipitates were collected as described above. Samples were heated at 95°C for 5 min with 1× sample buffer (NuPage LDS sample buffer, Invitrogen) with NuPage antioxidant (Invitrogen). Proteins were separated on 4–12% gradient NuPage bis–tris precast gels (Invitrogen) in MES–SDS running buffer (Formedium). Proteins were blotted onto nitrocellulose membranes (Amersham) at a constant current of 400 mA for 1 h. Membranes were blocked in 5% skimmed milk in PBST for 1 h at room temperature and incubated with primary antibodies (EMI2—Everest Biotech EB0606, Flag tag—Sigma F1804, p44/42 MAPK—Cell Signalling 91069, tubulin α—AbD Serotec MC178G) at 4°C overnight with secondary antibodies (Vector Laboratories PI-9500, GE Healthcare NA93 and NA934, Santa Cruz Biotechnology SC-2032) for 1–2 h at room temperature. The detection was performed, using ECL Prime or ECL Advanced (Amersham) and Kodak X-Ray films.