Ab6106

Overlap between eCLIP datasets A and B was determined by calculating the fraction of significant and reproducible peaks in dataset A that overlapped (by at least one base) a peak in dataset B, and vice versa the fraction of peaks in B that overlapped a peak in A, and taking the maximum of those fractions as the overall pairwise fraction overlap. Only datasets with at least 100 reproducible and significant peaks were used for this analysis. Gene Set Enrichment Analysis was performed using the GSEA software package [77 (link)]. RBP interaction data was obtained from the BioPlex 2.0 dataset [52 (link)].
IP-western validation was performed using HNNRPL (ab6106, Abcam), RBFOX2 (A300-864A, Bethyl), FMR1 (RN016P, Bethyl), AGGF1 (A303-634A, Bethyl), and TNRC6A (RN033P, MBLI) antibodies in UV crosslinked K562 cells. Immunoprecipitation in high-salt wash conditions was performed using standard eCLIP wash buffers, beads, and other reagents [18 (link)]. Low-salt co-immunoprecipitation conditions used identical conditions, except for lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% Sodium deoxycholate, and Protease Inhibitor cocktail (Promega)) and wash buffer (5 washes total in TBS + 0.05% NP-40). Westerns were probed with HNNRPL (ab6106, Abcam) primary antibody and TrueBlot secondary (Rockland).

The Abs used for immunoblotting were rabbit anti-PTBP1 Ab (382421, Zen Bioscience), rabbit anti-PSF Ab (15585-1-ap, Proteintech), rabbit anti-H3 Ab (17168-1-ap, Proteintech), mouse anti–hRNP L Ab (ab6106, Abcam), rabbit anti–hRNP U Ab (ab172608, Abcam), rabbit anti–hRNP A1 Ab (ab208026, Abcam), mouse anti–hRNP F Ab (04-1462, Sigma-Aldrich), and mouse anti-actin Ab (66009-1-Ig, Proteintech). The Abs used for co-IP were mouse anti-PTBP1 Ab (MABE986, Sigma-Aldrich) and mouse anti-PSF Ab (P2860, Sigma-Aldrich). The Abs used for RIP analysis were mouse anti-PTBP1 Ab (MABE986, Sigma-Aldrich), mouse anti-PSF Ab (P2860, Sigma-Aldrich), mouse anti–hRNP L Ab (ab6106, Abcam), rabbit anti–hRNP U Ab (ab172608, Abcam), rabbit anti–hRNP A1 Ab (ab208026, Abcam), mouse anti–hRNP F Ab (04-1462, Sigma-Aldrich), and mouse normal immunoglobulin G (IgG; sc-2025, Santa Cruz Biotechnology). The Abs used for IF were mouse anti-PTBP1 Ab (MABE986, Sigma-Aldrich), rabbit anti-PSF Ab (15585-1-AP, Proteintech), fluorescein isothiocyanate–conjugated donkey anti-mouse IgG (SA00003-9, Proteintech), and CoraLite 647–conjugated donkey anti-rabbit IgG (SA00014-7, Proteintech).

Western-Blot were performed as previously described20 (link)46 (link). Membranes were incubated overnight at 4 °C with the following antibodies: hnRNPL (Abcam ab6106, 1/2000), Galectin-3 (Abcam ab31707, 1/300) and β-actin (Sigma AC-15, 1/5000). The membranes were then incubated with peroxydase-conjugated secondary antibodies (Sigma-Aldrich) and revelation was performed with LAS 4000 (Fujifilm) using the West Pico chemoluminescent substrate (Perbio).