Fig 2: SILAC-based proteomics of FLNA-binding partner. (A) The CD cleft (blue) opens for partner interaction in force-dependent manner. FLNA domains used for affinity purification of mechanically-regulated FLNA-binding partner. IgFLNA1-2 was used as a negative control. (B) Schematic representation of the SILAC-based mass spectrometry experiments. (C) Standard scatterplots with normalized Log2 (H/L) ratios/Log10 Intensities (control versus test) highlighting the distribution of quantified proteins in each MS screening.

Fig 3: Schematic structure of Larp4 and identification of FLNA-binding domains on Larp4. (A) Structure of Larp4. RRM: RNA Recognition Motif. Blue indicates fragments of Larp4 that bind to FLNA R21. (B) CBB stained gel of 5 µg of the purified GST fusion protein. (C) GST-Larp4 fragments incubated with 2 µM purified His-eGFP-FLNA R21-22. Bound protein was detected by western blotting using rabbit anti-GFP antibodies.

Fig 4: Identification of critical amino acids of LARP4 for FLNA interaction. (A) Alignment of binding interfaces of FLNA-binding partners. Point mutations of the underlined amino acids of mouse Larp4 (F273) and human LARP4 (F277) to Ala were predicted to disrupt the interaction with FLNA R21. (B) GFP-LARP4 (Larp4) (WT, F277A in LARP4 and F273A in Larp4) were expressed in HEK293A cells. Cell lysate were mixed with GST-His-FLNA R21-22 immobilized on glutathione beads. CBB staining showed 2 µg of the purified Gst-His-FLNA R21-22 protein loaded to the 15% SDS-PAGE gel. Bound GFP-LARP4(Larp4) was detected by western blotting using anti-GFP antibodies.

Fig 5: Interaction of LARP4 with FLNA in living cells. (A) Approximately 1.2 × 106 HEK293A, Hela, hsSKM, and MEF cells cells were analyzed by WB using anti-LARP4 antibodies. The lower band is the indicative of the degradation of LARP4 protein. GAPDH is used as a loading control. (B) Co-localization of LARP4 and FLNA in HEK293A (Bar: 100 µm). Enlarged images are shown in the dotted box in white. Bar: 50 µm. White arrows indicate the cell-cell junction. (C) Interactions of LARP4 with FLNA R22 in HEK293A cells visualized by proximity ligation assay (PLA). Representative PLA images where the PLA signal (red) represents close proximity (<40 nm) between two proteins. PLA signal is significantly decreased when cells are treated with 5 µM Latrunculin B for 1 h. Graph shows the quantification of PLA between FLNA R1 and R22, R22 and R23, LARP4, LARP4 and R22 and under Lat B treatment respectively (n = 5). The nucleus was stained by Hoechst (blue). Scale bars are 20 µm ****p < 0.0001 was determined by the two-tailed unpaired Student’s test. (D) FRAP analysis was performed in HEK293A cells transfected GFP-LARP4 WT or GFP-LARP4 F277A. Quantitative analyses of FRAP assay results. Curves depict mean values (±SD) from measurements of at least 6 representative cells (n = 10). WT (blue), non-FLN-binding F277A (red). (E) Fmax, which represents the mobile fraction of the molecule in the bleached region, and t½, which is the time to recover half of the maximum fluorescence and is inversely correlated to the diffusion coefficient were analyzed by one-phase association model plugged into the Prism software. (95% confidence intervals).