Fig 2: FUS isoforms are differentially recognized by N- and C-terminal antibodies. Western blot analysis of whole-cell lysates of U87 cells transfected with FUS siRNA. FUS immunoreactive bands were blotted with antibodies recognizing either an (A) N- terminal (1–50 aa) or (B) C-terminal (500–526 aa) epitope. FL: Full-length FUS; NTF: N-terminal fragment; CTF (17): C-terminal fragment at 17 kDa; CTFs (25–50): C-terminal fragments distributed from 25 to 50 kDa; ns: non-specific band. Uncut nitrocellulose membrane was first blotted with anti-FUS antibody and HRP-conjugated secondary antibody followed by film exposure. The same membrane was then washed with 0.1% TBST and incubated with anti-GAPDH antibody. GAPDH bands were acquired using Bio-Rad ChemiDoc MP imaging system. Original blots are shown in the Expanded file 2.

Fig 3: Fluorescent labeling of mutant EC12 peptide.(A) EC12WT and mutant EC12 show similar binding capacity to Cry1Ab. Binding reactions of the EC12 peptides to Cry1Ab were conducted at room temperature for 15 min. Indicated samples were resolved on a native gel and silver stained afterwards.(B) Reaction showing the irreversible labeling of the cysteine residue on EC1236C/44W. “Dye” refers to Alexa-488, a fluorescent dye, which is conjugated to the maleimide moiety. Maleimide crosslinks the free thiol group depicted on the EC12 peptide.(C) Separation of Alexa-488 labeled EC1236C/44W from free dye molecules by size exclusion chromatography. The peak protein fractions were resolved by SDS-PAGE. Stain-free images (top panel) were captured using a ChemiDoc MP Imaging system (Bio-Rad) and fluorescent images (bottom panel) recorded using a Typhoon FLA 9500 fluorescent scanner (GE Healthcare Life Science).

Fig 4: Characterizing the GTP-Rac1, F/G actin levels in MOCK and T2R14 KD OKF6 cells. (A) The MOCK and T2R14 KD OKF6 cell lysates (500 µg) were incubated with PAK1 agarose beads (20 µg) for 1 h at 4 °C. After incubation the beads were washed, re-suspended in 2× Laemmli buffer and separated by 12% SDS-PAGE. Western blot analysis of PAK1 pull-down and total protein lysates using primary mouse monoclonal anti-Rac1 antibody (1:1000), mouse monoclonal anti ß-Actin antibody (1:25,000) and secondary anti-mouse HRP antibody (1:5000 and 1:25,000) re-spectively. To visualize GTP-Rac1 that is at low levels compared to Total-Rac1, the blots were exposed for 5 min. To visualize PAK1 associated Actin and ß-Actin the blots were exposed for 1 min. (B) Western blot analysis of F and G actin fractions isolated from MOCK and T2R14 KD cells and separated by 10% SDS-PAGE. The blots were probed using primary mouse monoclonal anti ß-Actin antibody and exposed for 1 min. (C) The corresponding densitometry analysis of F and G actin isoforms. The data represented in the graphs are SEM of =3 independent experiments. One-way ANOVA analysis, using Tukey’s multiple comparison analysis, was performed, and the observed p-value is * p < 0.05. The blots were developed by using chemiluminescence detection system and imaged by using a ChemiDoc MP imaging system, Bio-Rad, Toronto, ON, Canada).

Fig 5: Aggregation of MUC5B protein domains by EGCG.(A) Rate-zonal centrifugation of 380 µg/mL NT5B recombinant protein (D1-D2-D'-D3 domains of MUC5B) in PBS (black circles) or 4 mM EGCG (grey triangles) in 5–20% (w/v) sucrose gradients (n = 2). Sucrose gradients were fractionated into 21 fractions and pelleted material was solubilised in urea. Fractions were run on SDS-PAGE gels and silver stained. NT5B is expressed as a monomer and a dimer. Inset (i) shows the SDS-PAGE gel of the first 11 fractions of NT5B in PBS sucrose gradient, showing all NT5B was contained within the first 11 fractions. Inset (ii) shows the SDS-PAGE gel of fractions 12–21 and the pellet of an NT5B with EGCG gradient, showing that in the presence of EGCG all NT5B is pelleted to the bottom of the tube. (B) Rate-zonal centrifugation of 500 µg/mL CT5B recombinant protein (D4-B-C-CK domains of MUC5B) in PBS (black circles) or 4 mM EGCG (grey triangles) in 5–20% (w/v) sucrose gradients (n = 2). Gradients were fractionated into 19 fractions and pelleted material was solubilised in urea. Fractions were run on SDS-PAGE gels and Coomassie stained. CT5B is expressed as a monomer and a dimer. Inset (i) shows the SDS-PAGE of the first 10 fractions of a sucrose gradient of CT5B in PBS, showing all CT5B was present in the first 5 fractions. Insets (ii) and (iii) show the SDS-PAGE gels of fractions 1–10, and 11–19 and the pellet, respectively, showing that in the presence of EGCG, there is faster sedimentation of CT5B but not full aggregation. Stained SDS-PAGE gels were imaged on a Biorad ChemiDoc MP Imaging System to measure band intensities. Pel. = pelleted material.