α β tubulin

Cells were lysed with RIPA buffer (Thermo Fisher) containing protease inhibitor cocktails (Roche) and stored at −80°C. Protein concentration was determined using the BCA protein assay kit (Thermo Fisher) according to the manufacturer instructions. 20 µg of total protein were loaded on a NUPAGE 4–12% Bis-Tris polyacrylamide gel (Thermo Fisher) and transferred to PVDF membranes (iBlot, Thermo Fisher). The membranes were blocked with TBS-0.1% Tween20, 5% non-fat dry milk for 30 min at RT and incubated overnight with primary antibodies against α-β-Tubulin, p-62 (Cell Signaling, Danvers, Massachusetts) and LC3 (Abcam, Cambridge, United Kingdom). Membranes were washed in TBS-Tween and incubated with secondary HRP-conjugated antibody (GE Healthcare, Chicago, IL) at RT for 1 hr. Membranes were washed and exposed to SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher). Detection and quantification of band intensities was performed using Azure Imager C400 (Azure Biosystems, Dublin, CA) and ImageJ software (version 1.51).

Respiratory epithelial cells on glass slides, stored at -80°C, were defrosted, washed with PBS and fixed by incubating with 4% PFA for 15 min. The samples were then treated with 0.2% Triton-X 100 for 10 min. to permeabilize the cell membranes, and incubated with 1% skim milk at 4°C overnight to block unspecific binding of the antibodies. Prepared samples were incubated with the primary antibodies for 2–3 h at room temperature, washed and incubated with the cross-absorbed secondary antibodies for 25 min at room temperature, and then with Hoechst 33342 (Sigma). The primary antibodies were IgGs directed against markers of various elements of the ciliary ultrastructure: acetylated α-tubulin (mouse monoclonal, SIGMA); α/β-tubulin (rabbit polyclonal, Cell Signaling Technology); DNAH5 (mouse monoclonal, generated [10 (link)]); DNALI1 (rabbit polyclonal, AVIVA); LRRC6 (rabbit polyclonal, AVIVA); CCDC39 (rabbit polyclonal, SIGMA). Immunofluorescence images were taken with Olympus Bx41 Microscope and processed with Isis software Metasystem International) and Adobe Creative Suite 4.

The antibodies used for immunoblot analysis were CHD1 (Bethyl Laboratories), CHD2 (Cell Signaling), α/β-Tubulin (Cell Signaling) and Alexa Fluor 680 donkey anti-rabbit IgG (Invitrogen). 48 h post-transfection cells were lysed in 2x NuPAGE LDS sample buffer (Invitrogen) and resolved by SDS-PAGE on NuPAGE 4-12% Bis-Tris gels (Invitrogen). Proteins were transferred to Immobilon-FL PVDF membranes (Millipore) and membranes were for blocked for 1 h in 0.1% Casein/0.2x PBS (Biorad). The membrane was then probed with primary antibody in blocking buffer +0.1% Tween-20 overnight at 4°C. Membranes were washed three times with PBS +0.1% Tween-20, then incubated with Alexa Fluor 680 donkey anti-rabbit secondary antibody in blocking buffer +0.1% Tween-20 + 0.01% SDS for 1 h at room temperature. Immunoblots were washed, then visualized and quantitated using an Odyssey infrared imaging system (Licor). CHD1 and CHD2 protein levels were normalized to α/β-Tubulin levels.

Western blots were performed as previously described (Chung et al., 2016 (link)). Briefly, proteins were extracted from cell pallets with RIPA buffer (Sigma, cat# R0278) containing protease inhibitor (Roche, cat# 04693159001). The concentrations of extracted cellular proteins were measured using a QuantiPro BCA assay kit (Sigma-Aldrich, cat# QPBCA). Equal amounts of protein were loaded into NuPage Bis-Tris gels (Life Technologies, cat# NP3023BOX) and transferred to a polyvinylidene difluoride membrane with iBlot semi-dry transfer system (Invitrogen, cat# IB21001). The membranes were blocked with 5% BSA in TBST and primary antibody was incubated overnight at 4°C. After incubation with secondary antibodies conjugated with horseradish peroxidase, protein bands were visualized using the G:Box BioImaging systems and quantified using the GeneTools image scanning and analysis package. Protein expression was normalized to α/β-tubulin (rabbit polyclonal, 1:2000; Cell Signaling #2148), which serves as a loading control.

Ginkgolic Acid [(GA,15:1) - CAS 22910-60-7, 345887] was purchased from Millipore. N-ethylmaleimide (NEM, SENPs inhibitor, E1271), streptonigrin (SN, SENPs inhibitor, S1014) were bought from Sigma. Protein A/G PLUS-Agarose (sc-2003) was obtained from Santa Cruz.
Antibodies for immunoblotting, immunoprecipitation, and immunostaining were: V5 (Cell Signaling Technology, 13202, Rabbit, WB 1:1000), Trx2 (Abcam, ab185544, Rabbit, WB, 1:10000), Trx2 (Santa Cruz, sc-133201, Mouse, IF, 1:50), Phospho-p53 (Cell Signaling Technology, 9284, Rabbit, WB 1:500), P21 (Cell Signaling Technology, 2947, Rabbit, WB 1:1000), Phospho-Histone H2A.X (Cell Signaling Technology, 9718, Rabbit, WB 1:200), β-Actin (Cell Signaling Technology, 4970, Rabbit, WB 1:1000), α/β-Tubulin (Cell Signaling Technology, 2148, Rabbit, WB 1:1000), TFAM (Cell Signaling Technology, 8076, Rabbit, IF 1:100), PMPCA (Santa Cruz, sc-390471, mouse, IP 1:50 WB 1:500), SUMO1 (Cell Signaling Technology, 4930, Rabbit, WB 1:200), SUMO2/3 (life technologies, 519100, Rabbit, 1:600), Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-21202, IF 1:200), Donkey anti-Rabbit IgG (H + L) Highly Cross- Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher Scientific, A-21207, IF 1:200), normal mouse IgG (Santa Cruz, sc-2025, IP 1:100).

Cells were lysed with Laemmli buffer consisting of 8.0% glycerol, 3% sodium dodecyl sulfate (SDS) and 200 mM Tris–HCl (pH 6.8), followed by boiling at 100°C for 5 min. Protein concentrations were measured by a DC™ protein assay kit (Bio-Rad; Cat. No.: 5000111) according to the manufacturer's instructions. Equal amounts of proteins were loaded and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Afterwards, the resolved proteins were transferred onto 45-μm polyvinylidene difluoride (PVDF) membrane (Merck Millipore; Cat. No.: IPVH00010). Next, 5% non-fat dry milk dissolved in Tris-buffered saline with 0.1% Tween 20 (TBST) was used to block the membrane at RT for 1 h. Membranes were incubated overnight at 4°C with the respective primary antibodies recognizing S. pyogenes Cas9 (Abcam; Cat. No.: ab191468), α/β Tubulin (Cell Signaling; Cat. No.: 2148), and GAPDH (Merck Millipore; Cat. No.: MAB374) diluted 1:1000 in TBST supplemented with 5% BSA. Subsequently, the membranes were washed with TBST thrice and probed with secondary antibodies specific for mouse IgG (Sigma-Aldrich; Cat. No.: NA931V) or rabbit IgG (Cell Signaling; Cat. No.: 7074S) diluted 1:5000 in TBST containing 1% non-fat dry milk at RT for 2 h. Clarity™ Western ECL Substrate (Bio-Rad; Cat. No.: 1705060) was applied for signal detection using the ChemiDoc Imaging System (Bio-Rad; Cat. No.: 17001402).