Hnrnpa1

Protein extraction and Western blot experiments were performed as described previously [37 (link)]. Primary antibodies against the following antigens were used: PTBP1, hnRNPA1, and LC3B (Cell Signaling Technology, Danvers, MA, USA); PKM1 and PKM2 (Novus Biologicals, Littleton, CO, USA); SRSF3 (SRp20) (MBL, Nagoya, Japan). The 2nd antibodies used were HRP-conjugated horse anti-mouse or goat anti-rabbit IgG antibody (Cell Signaling Technology). The quantity loaded was verified by re-incubating the same membrane with anti-β-actin antibody (Sigma-Aldrich) or anti-GAPDH (Cell Signaling Technology).

For immunocytochemistry, cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature (RT). Cells were permeabilised and nonspecific antibody binding was blocked using 0.3% Triton-X containing 5% bovine serum albumin (BSA) (Sigma) in PBS for 60 min. Primary antibodies used were SMI-32 (BioLegend, San Diego, CA, USA; 801,701; mouse; 1:1000), ChAT (Millipore, Burlington, MA, USA; AB144P; goat; 1:100), β-III-tubulin (abcam, Cambridge, UK; ab41489; chicken; 1:1000), TDP-43 (ProteinTech, Rosemont, IL, USA; 12892-1-AP; rabbit; 1:400), SFPQ (abcam, Cambridge, UK; ab11825; mouse; 1:400), FUS (Santa Cruz, Santa Cruz, CA, USA; sc-47711; mouse; 1:200), hnRNPA1 (Cell Signaling, Danvers, MA, USA; 8443S; rabbit; 1:500) and hnRNPK (Santa Cruz, Santa Cruz, CA, USA; sc-28380; mouse; 1:500). Primary antibodies were made up in 5% BSA and then applied overnight at 4 °C. Cells were washed with PBS 2 times, and secondary antibody was added using a species-specific Alexa Fluor-conjugated secondary antibody (Life Technologies, Carlsbad, CA, US) at 1:1000 dilution in 5% BSA for 90 min at RT in the dark. Cells were washed once in PBS containing 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (1:1000) for 10 min. Following a second wash in PBS, cells were left in PBS in the dark.

Radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China) was used to extract total protein. The protein concentration was measured by a BCA protein assay kit (Thermo Fischer Scientific, Waltham, MA, USA). Equivalent proteins were electrophoresed via 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. After membranes were blocked in blocking buffer, it was followed by incubation with primary antibody at 4 °C overnight targeting hnRNPA1 (1:1000; Cell Signaling Technology, Beverly, MA, USA), Trim21 (1:1000; Cell Signaling Technology), PKM2 (1:1000; Cell Signaling Technology), PKM1 (1:1000; Cell Signaling Technology), IGF2BP3 (1:5000, Abcam, Cambridge, MA, USA), and GAPDH (1:3000; Cell Signaling Technology). Secondary antibody HRP-labeled rabbit IgG (1:5000; Cell Signaling Technology) was probed for 1 h at room temperature. The bands were detected by enhanced chemiluminescence.

Whole cell extracts of cultured cells were prepared in radioimmunoprecipitation (RIPA) lysis buffer supplemented with phosphatase and protease inhibitors (Calbiochem, Burlington, MA, USA). Protein concentration was determined by the bicinchoninic acid assay (BCA) (Thermo Fisher Scientific, Waltham, MA, USA) and separated by SDS-PAGE gel. The following antibodies and dilutions were used: hnRNPA1 (1:2000, Cell Signaling, Danvers, MA, USA), cleaved PARP1 (1:1000, Cell Signaling), HSP90 (1:4000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), GAPDH (1:3000, EMD Millipore, Billerica, MA, USA), Survivin (1:2000, Cell Signaling), and Flag-tag (1:2000, Sigma-Aldrich). Following blocking with 5% BSA, membranes were incubated with the primary antibodies overnight at 4 °C. The next day, membrane was incubated with secondary antibodies for 1 h at room temperature. Secondary anti-mouse IgG (A4416) and anti-rabbit IgG (A6667) antibodies were purchased from Sigma-Aldrich and used at a 1:4000 dilution. Images of blots were acquired on HyBlot ES Autoradiography Film (Thomas Scientific, Swedesboro, NJ, USA) following incubation with SuperSignal West Pico PLUS (Thermo Fisher Scientific). When necessary, membranes were stripped using Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) according to manufacturer’s instructions.

Immunoblotting was performed as described previously^{57 (link)}. Briefly, whole-cell lysates were prepared using RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Roche). Proteins were separated by running on 10% SDS-PAGE gels and transferred to PVDF membranes. After blocking with 5% skim milk in TBST, membranes were probed with appropriate primary antibodies overnight and further incubated with goat anti-rabbit IgG-HRP (Proteintech). Blots were developed using Western chemiluminescent HRP substrate (Millipore) and visualized using an imaging system (Tanon, 5200). The primary antibodies used in immunoblotting are as follows: β-actin (1:2000, Proteintech, 20536-1-AP), hnRNP F (1:1000, Thermo, PA522341), hnRNPA1 (1:1000, Cell Signaling Technology, #8443), hnRNPA2B1 (1:1000, ABclonal, A1162), p-IKKα/IKKβ (1:1000, Cell Signaling Technology, #2078), IKKβ (1:1000, Cell Signaling Technology, #8943), p-IKBα (1:1000, Cell Signaling Technology, #2859) and IKBα (1:1000, Cell Signaling Technology, #9242).