Gel transfer stacks nitrocellulose membranes

Western blot analyses were performed as previously described [26 (link)]. Briefly, cells were lyzed in RIPA 1X buffer (Sigma®) containing protease inhibitors (Sigma®) and phosphatase inhibitors (Roche®). Proteins were quantified by Pierce BCA Protein Assay kit (Pierce®) using a multiplate colourimetric reader, CLARIOstar (BMG Labtech®). Protein extracts (20 to 40 μg) were loaded on a 4%–12% SDS‐PAGE gradient (NuPage Bis‐Tris gels, Invitrogen®) and transferred onto Gel Transfer Stacks Nitrocellulose membranes (Invitrogen®) using the iBlot2 Dry Blotting System (Invitrogen®). Membranes were then incubated overnight at 4°C with the following primary antibodies: MBNL1 ‐ MB1a(4A8) (DSHB®, 1:1000) and MBNL2 ‐ MB2a(3B4) (DSHB®, 1:1000). After hybridisation of the peroxidase‐conjugated secondary antibody (from 1:50000 to 1:10000), immunoreactive bands were revealed by using Amersham ECL Select Western Blotting Detection Reagents (GE Healthcare®). Equal protein loading was verified by the detection of β‐Actin using the A3854 Monoclonal Anti‐β‐Actin−Peroxidase antibody (Sigma®, 1:10000).

Western blots analyses were performed as previously described (59 (link)). Briefly, cells were lyzed in RIPA 1× buffer (Sigma®) containing protease inhibitors (Sigma®) and phosphatase inhibitors (Roche®). Proteins were quantified by Pierce BCA Protein Assay kit (Pierce®). Protein extracts (15–20 μg) were loaded on a 4–12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis gradient (NuPage Bis–Tris gels, Invitrogen®) and transferred onto Gel Transfer Stacks Nitrocellulose membranes (Invitrogen®) using the iBlot2 Dry Blotting System (Invitrogen®). Membranes were then incubated overnight at 4°C with the following primary antibodies: MBNL1–MB1a(4A8) (DSHB®, 1:1000), MBNL2–MB2a(3B4) (DSHB®, 1:1000), MBNL3–5A11(sc-136 168) (Santa Cruz, 1:100). After hybridization of the peroxidase-conjugated secondary antibody (1:10000), immunoreactive bands were revealed by using Amersham ECL Select Western Blotting Detection Reagents (GE Healthcare®). Equal protein loading was verified by the detection of β-actin using the A3854 Monoclonal Anti-β-Actin−Peroxidase antibody (Sigma®, 1:10000).

Protein extracts (5–10μg) were loaded on a 3–8% (NuPage Tris-Acetate gels, Invitrogen®) or 10% (NuPage Bis–Tris gels, Invitrogen®) gels and transferred onto Gel Transfer Stacks Nitrocellulose membranes (Invitrogen®) using the iBlot2 Dry Blotting System (Invitrogen®). Antibody binding was quantified using a LiCor Odyssee CLx imager and Image Studio Lite 5.2 software. All antibodies used are listed in the Supplementary Material.