Raw Data Files Associated with Figure 2 in RBL2 represses the transcriptional activity of Multicilin to inhibit multiciliogenesis.

Immunoprecipitation, immunoblotting analysis, and silver staining
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.