Rabbit anti alpha tubulin

Wild‐type virgin female flies (w¹¹¹⁸) were aged either for 3, 15, 30, and 45 days or for 1–5 weeks as indicated in Results. Three thoraxes were dissected and homogenized in 1x Laemmli buffer. Immunoblots were performed as described in (Vrailas‐Mortimer et al., 2011 (link)). Membranes were developed using SuperSignal West Femto kit (ThermoFisher) or Pierce ECL (ThermoFisher) and exposed on autoradiography film. Antibodies used were: rabbit anti‐GFP 1:1000 (Invitrogen), rabbit anti‐starvin 1:10,000 (gift of Jrög Höhfeld), mouse anti‐actin 1:5,000,000 (Sigma), mouse anti‐FLAG M2 (Sigma), rabbit anti‐alpha tubulin (Cell Signaling Technologies), mouse anti‐Lamin 1:100 (DHSB), rabbit anti‐phospho‐Lamin A Ser22 1:1000 (Thermofisher), mouse anti‐beta tubulin (E‐10) 1:5000 (Santa Cruz Biotechnology), mouse anti‐ HRP 1:20,000 (Jackson Labs), rabbit anti‐HRP 1:40,000 (Jackson Labs). Densitometry was performed using a minimum of three independent blots. For statistical analysis of protein expression level, pixel density of the tested protein was normalized within sample to the loading control. These values were then normalized to control to calculate fold change. The fold change values were analyzed by Student's t‐test or ANOVA (R. C. Team, 2015 ) as appropriate.

HUASMCs were lysed using sample buffer containing 1% Triton X-100 and 0.1 μmol/L DTT or buffers for preparing nuclear and cytosolic fractions. Protein samples were separated by SDS (10%), blotted onto nitrocellulose membranes and analyzed by chemiluminescence-based immunodetection according to standard procedures. Primary antibodies: rabbit anti-NFAT5 1:500 (Santa Cruz Biotechnology), rabbit anti-ACTBL2 (κ-actin) 1:1000 (abcam), mouse anti-β-actin 1:10,000 (abcam), rabbit anti-histone H3 1:1000 (abcam), rabbit anti-alpha-Tubulin (Cell Signaling) 1:1000.

Primary human arterial endothelial cells and PASMC were lysed in RIPA buffer (Sigma-Aldrich) and 10 μg of protein were run on a 10% SDS polyacrylamide gel, followed by electrotransfer to an Amersham Hybond P 0.45 PVDF Blotting Membrane (GE Healthcare, Wien, Austria). After blocking with 5% non-fat dry milk in TBS-T buffer (Tris-buffered saline with 0.1% Tween-20), the membrane was incubated overnight at 4°C with one of the following antibodies: anti-p38, anti–phospho-p38 (T180/Y182), anti-c-Jun; anti-phospho c-Jun (S73), anti-phospho Erk1/2 (T202/Y204), anti-Erk1/2, anti-phospho c-Fos (S32) all from Cell Signaling and anti-c-Fos (Novus Biologicals, Cambridge, UK) were used at dilution of 1:1000; and anti-TLR4 (Abcam), anti-RAGE (1:500; Abcam) and rabbit anti–alpha-tubulin (1:5000; Cell Signaling, Boston, MA, USA). After washing, the membranes were incubated with horseradish-peroxidase–labelled secondary antibodies (1:5000, anti-rabbit-HRP, Pierce, Thermo Fisher Scientific, Waltham, MA, USA) and proteins detected with Amersham ECL Prime Western Blotting Detection System (GE Healthcare). Antibodies were removed by incubating the membrane for 15 min. with stripping buffer (RestoreTM PLUS Western Blot Stripping Buffer; Thermo Scientific). Densitometric analysis was performed with ImageJ (National Institutes of Health, USA).