Srsf1

Co-immunoprecipitation (co-IP) using PANC-1 cell lysate and antibody of SRSF1 (Santa Cruz, sc-33,652), EIF3B (Santa Cruz, sc-137,214), TIA1 (Santa Cruz, sc-166,247), GAPDH (ProMab, 20,035), and IgG (Santa Cruz sc-2025) was performed as described previously [33 (link)], and the protein complex was detected by western blot.

Cells were lysed in Laemmli buffer and analyzed for total protein concentration. 20 μg of total protein from each cell lysate was separated by SDS-PAGE and transferred on to a PVDF membrane. The membranes were blocked with 5% milk and probed with specific antibodies. Bands were visualized using enhanced chemiluminescence detection. Primary antibodies: β-Catenin (1:2000, Sigma), Phospho-MEK S217/221 (1:1000, Cell Signaling), total MEK (1:1000, Cell Signaling), phospho-ERK T202/Y204 (1:1000 Sigma), total ERK1/2 (1:1000, Cell Signaling), phospho-AKT (1:1000, Cell Signaling), AKT (1:1000, Cell Signaling), cleaved Caspase 3 (1:1000, Cell Signaling) SRSF1 (AK96 culture supernatant 1:300), CUGBP1 (1:1000, Santa Cruz Biotechnology), MBNL1 (1:250, Santa Cruz Biotechnology), SRSF3 (1:1000, Santa Cruz Biotechnology). Secondary antibodies: HRP-conjugated goat anti-mouse, goat anti-rabbit and donkey anti-goat IgG (H+L; 1:10000 Jackson Laboratories).

Equal amounts (10–30 mg) of protein extracts from cerebral tissues or cells were loaded and separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) using 8–12% acrylamide gradients. Following electrophoresis, separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Piscataway, NJ, USA). Non-specific proteins were blocked by incubating the membrane in blocking buffer (5% non-fat dry milk in T-TBS containing 0.05% Tween 20) overnight. The membranes were incubated with the indicated primary antibodies against Pnn (P3A, 1:1000, a kind gift from Prof. Pin Ouyang), SRSF1 (1:500, Santa Cruz, Santa Cruz, CA, USA), SRSF2 (1:500, Santa Cruz, Santa Cruz, CA, USA), GR (1:1000, Abcam, Cambridge, MA, USA), GPx (1:500, Abcam, Cambridge, MA, USA), HO-1 (1:1000, Sigma, St. Louis, MO, USA), NQO-1 (1:500, Abcam, Cambridge, MA, USA), NOX-1 (1:2000, St. Louis, MO, USA), NOX-2 (1:1000, St. Louis, MO, USA), p21 (1:500, Santa Cruz, CA, USA), TGF-β (1:500, Abcam, Cambridge, MA, USA), and actin (1:1000, Cell Signaling, Danvers, MA, USA) for 1 h at room temperature. Signals were detected with HRP-conjugated goat antimouse or goat ant-rabbit with ECL (Perkin Elmer, Waltham, MA, USA).

Murine palatal shelf tissues of E13.5, E14.5, and E16.5 were dewaxed in xylene, and then dehydrated in ethanol. The tissues were then incubated in solutions containing specific primary antibodies: EIF3H (1:100, Santa Cruz, sc-271283), SRSF1 (1:100, Santa Cruz, sc-33652), and RBBP6 (1:100, Santa Cruz, sc-9962) (Table S3) followed by incubation in Alexa fluor 488-labeled goat anti-mouse secondary antibodies (1: 50, Beyotime, A0428).

Hematoxylin and eosin (H&E) staining was performed according to the manufacturer’s instructions. In brief, human sample tissues of NSCL/P were immersed in 4% paraformaldehyde for 48 h. Next, the fixed tissues were dehydrated, cleared, and embedded in paraffin wax. The paraffin blocks were cut into 4-μm-thick sections and stained with hematoxylin and eosin (H&E). Primary antibodies against the following proteins were used: EIF3H (1:100, Santa Cruz, sc-271283), SRSF1 (1:100, Santa Cruz, sc-33652), and RBBP6 (1:100, Santa Cruz, sc-9962) (Table S3). Briefly, tissues were fixed, dehydrated, embedded, and sectioned for each sample for all stains. Sections were incubated with primary antibodies, washed, and then incubated with appropriate secondary antibodies (MaxVision, kit-5020, China). Optical microscopy (Thermo Scientific, Wilmington, USA) was used to image stained sections. Semi-quantitative analysis was performed using Fiji v2.9.0 (NIH, Bethesda).