Ab21679

72 h after transfection, cells were fixed for 4 min in methanol cooled to −20 °C. Thereafter, samples were typically blocked in phosphate-buffered saline (PBS) containing 3% bovine serum albumin for 40 min at room temperature. Cells were then incubated with primary antibodies for 60–90 min, washed four times with PBS, labelled with Alexa Fluor-conjugated secondary antibodies (ThermoFisher Scientific) diluted 1:500 for 30–45 min, washed four times with PBS and mounted with ProLong Gold (Life Technologies). Cells were labelled with a mouse anti-CHC (610500, BD Biosciences) or a rabbit anti-CHC (ab21679, Abcam) antibody diluted 1:200 together with an Alexa Fluor 488-conjugated anti-α-tubulin antibody (322588, Life Technologies) and 1 μg/ml DAPI.

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.

The following primary antibodies were used in the present study: mouse anti-Ttyh1 (1:200, #WH0057348M4, Sigma), rabbit anti-Synaptotagmin 1 (1:100, #105102, Synaptic Systems), rabbit anti-Synaptoporin (1:500, #102002, Synaptic Systems), rabbit anti-Neuronal Class III β-Tubulin (1:750, #PRB-435P, Covance), rabbit anti-LAMP1 (1:500, #ab24170, Abcam), rabbit anti-GRP78 (1:200, #ab21685, Abcam), rabbit anti-Calnexin (1:40, #ab22595, Abcam), rabbit anti-GM130 (1:200, #ab52649, Abcam), rabbit anti-TGN46 (1:500, #ab16059), rabbit anti-Clathrin (1:500, #ab21679, Abcam), rabbit anti-Rab5 (1:500, #ab18211, Abcam), rabbit anti-Rab11 (1:100, #71-5300, Invitrogen), rabbit anti-APPL2 (home made, [20 (link)]), rabbit anti-Olig2 (1:500, #AB9610, Chemicon Millipore), rabbit anti-Iba1 (1:1,000, #019-19741, Wako), mouse anti-GFAP-Cy3 (1:1,000, #C9205, Sigma). Anti-IgG mouse (1:200, #553447, BD Pharmingen) anti-IgG rabbit (1:200, #ab46540, Abcam) were used as controls to test specificity of immunostainings. The following secondary antibodies were used in the present study: goat anti-mouse conjugated with Alexa Fluor 488 (1:200, #A11001, Molecular Probes), goat anti-rabbit conjugated with Texas Red (1:200, #T2767, Molecular Probes), horse anti-mouse conjugated with Texas Red (1:200, #TI2000, Vector Laboratories), and anti-mouse biotinylated antibody (1:200, #BA2001, Vector Laboratories).

Primary antibody against PCAF (3305, CST), p53 (sc-126, santa cruz), p21 (sc-6246, santa cruz), p16 (sc-166760, santa cruz), Ubiquitin (sc-53509, santa cruz), phosphor-Histone H2A.X(Ser139) (#2577, CST), YAP (#14074, CST), MDM2 (BS-1223, Biogot), phospho-YAP (#13008, CST), TAZ (#83669, CST), phospho-TAZ (#59971, CST), clathrin (ab21679, abcam), caveolae (ab2910, abcam), mTOR (ab2732, abcam), p-mTOR (Ser2448) (ab109268, abcam), p-mTOR(Ser2481) (ab137133, abcam), IL-1β (sc-12742, snata cruz), IL-6 (sc-130326, santa cruz), GAPDH (#AP0063, Biogot), histone H3(BS3718, Biogot), and PA (P0500, Sigma-Aldrich) were purchased commercially.

Anti-clathrin heavy chain antibody (ab21679), anti-dynamin 2 antibody (ab3457) were obtained from Abcam. Caveolin-1 polyclonal antibody (PA5-17447), Alexa Fluor 488 goat anti-rabbit IgG (A11008) and horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody (31458) were from Thermo Fisher Scientific.

HASM were cultured in SmGM2 at 37 °C under 5% CO₂. Cells of the passage number between 5 and 6 were used for experiments. Cells were rinsed with Dulbecco’s phosphate-buffered saline (PBS) once and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10 min at room temperature, followed by permeabilization with 0.3% TritonX-100 in PBS for 15 min or with 90% chilled methanol for 5 min. After blocking in 5% normal goat serum (FUJIFILM Wako Pure Chemical Corp.) and 0.3% TritonX-100 in PBS, cells were incubated with rabbit polyclonal anti-clathrin heavy chain (1:400) (#ab21679, Abcam) overnight at 4 °C. After washing, cells were incubated with an appropriate Alexa-Fluor-conjugated secondary antibody (Molecular Probes) for 1 h at room temperature.