DOI: 10.6084/m9.figshare.23792181
title: Raw data files associated with Figure 4 in RBL2 represses the transcriptional activity of Multicilin to inhibit multiciliogenesis.

description: <b>Immunoprecipitation, immunoblotting analysis, and silver staining</b>For identifying interacting proteins of FLAG-tagged Multicilin by MASS analysis, MEFs were infected by pAd/CMV/V5 vector encoding 3xFLAG-Multicilin, NLS-6xmyc-E2f4ΔCt-VP16-T2A-Multicilin, and NLS-6xmyc-E2f4WT-T2A-Multicilin. Non-infected MEFs were used as the negative control. Two days after infection, the non-infected MEFs or infected MEFs were lysed in lysis buffer (50mM HEPES-KOH [pH 7.5], 500NaCl, 5mM EDTA [pH 8.0]), 2% Triton, DNase I (10ug/ml)) by incubation on ice for 20min. Lysates were cleared by centrifugation at 12,000 rpm for 20 min at 4 °C, then precleared by incubation with Protein G agarose (Invitrogen #20397) agitating for 1hr at 4°C. Spun-down beads were incubated with FLAG M2 agarose (Sigma #A2220) agitating for 2.5 hours at 4°C. The beads were washed twice with wash buffer (1:1 mix of 50mM HEPES and Lysis buffer), washed with lysis buffer twice, then washed twice again with wash buffer. Spun-down beads were incubated with 40uL of FLAG peptide (500ng/mL) for bound protein elution overnight at 4°C. Elutes were subjected to protein sampling for further immunoblot analysis and silver staining. Eluted protein samples and experimental protein lysates were subjected to SDS-PAGE and transferred to a PVDF membrane that was then blocked with 0.5% casein blocker (#161-0783, Bio-Rad) in PBS for 30 min followed by incubation with the indicated primary antibodies overnight at 4 °C in 0.1% Tween-20 in blocking buffer. After extensive washing in 0.1% Tween-20 in PBS, the blots were incubated with Alexa 680 or 800-conjugated anti- mouse or rabbit secondary antibodies (Invitrogen, A21058, A21076, A32735) for 45 min at room temperature, washed with 0.1% Tween-20 in PBS, and imaged using Odyssey (LI-COR). Silver staining was performed with Pierce Silver Stain Kit (Thermo #24612) based on the manufacturer’s instructions.<b>Immunocytochemistry and image processing</b>MEF centriolar and cilia imaging was performed as previously described<sup>10</sup>. HBECs were grown on Corning 6.5mm Transwell inserts (#3470) or Nunc Lab-Tek chamber slides (#177445) and fixed in 4% paraformaldehyde in PBS overnight at 4°C. Cells were concurrently permeabilized and blocked-in antibody dilution buffer (5% bovine serum albumin, 0.3% Triton X-100, PBS) before incubation with antibodies (<b>Supplemental Table S4</b>) in antibody dilution buffer. Samples were incubated at room temperature with primary antibodies for 2 hours and secondary antibodies for 45 minutes. Counterstains in PBS were performed directly after secondary antibody incubation (DAPI 1µg/mL 5 minutes, Phalloidin (Biolegend#424203) 5 units/mL), with (3x) 5-minute PBS washes performed after primary antibody and counterstain incubations. Samples were mounted with No. 1.5 coverslips using Fluoromount-G mounting medium (Invitrogen #00-4958-02).Fluorescent image tile scans were performed on either the DMi8 (Leica) or Revolution (Echo) fluorescent microscope systems and processed in Fiji<sup>38</sup>. TP73 positive cells were quantified by thresholding for positive DAPI and TP73 signal based on control samples, water shedding and then analyzing particle counts to give the number of nuclei positive for each stain. Cilia area coverage was calculated by thresholding for positive acetylated α-tubulin staining and then dividing by the area covered by counterstain (Phalloidin), with a thresh hold set high enough to give positive signal for the entire cell coverage area.