N cad

Cellular lysates were prepared in RIPA (Boston Bioproducts, Ashland, MA, USA) containing Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, Grand Island, NY 14072 USA). Protein was quantified using the BCA assay kit (Thermo Scientific, Grand Island, NY 14072), separated on SDS-PAGE gels and transferred to the Immun-Blot® PVDF membrane (Bio-Rad, Hercules, California 94547USA). Membranes were blocked in 5% nonfat milk and incubated with the following primary antibodies; BMI1 (Invitrogen at 1:1000 dilution), Fibronectin, N-Cad, E-Cad (BD Biosciences at 1:1000) EMA (Dako at 1:500 dilution), EPCAM, Snail, (Cell signaling technology at 1:1000 dilution), ATP7B (Protein Tech at 1:500) TWIST1, α tubulin and β actin (Sigma, TWIST1 at 1:1000, α tubulin and β actin at 1:10,000 dilutions), Secondary antibodies (from Sigma) were used at a concentration of 1:10,000.

The following primary antibodies were used in this study: GAPDH, ZEB1, E-Cad, FoxM1, HELLS, Cyclin B1, Cyclin D1, Cyclin A, K14, β-catenin, ZEB1, BrdU, and p63 from Santa Cruz Biotech; GRHL2 (Abnova, Taipei City, Taiwan; H00079977-A01); N-Cad from BD Biosciences (San Jose, CA); FN and Snail from Sigma-Aldrich; p-Smad3 (ser423/425), p-Smad2 (ser465/467), Smad4, p-p38 (Thr180/Tyr182), p-Erk1/2 (Thr202/Tyr204), p-JNK (Thr183/Tyr185), p-p65 (Ser536), and Oct-4 from Cell Signaling Technology Inc. (Danvers, MA); hTERT and Sox2 from Abcam (Cambridge, MA); TGF-β from Novus (Littleton, CO); and PCNA from Calbiochem (San Diego, CA). Secondary peroxidase-conjugated anti-rabbit or anti-mouse antibodies were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Tmx and 4-NQO were purchased from Sigma-Aldrich, while TGF-β1 was from PeproTech Inc. (Rocky Hill, NJ). JNK selective inhibitor SP600125 was purchased from Sigma-Aldrich and MEk1/2 inhibitors U0126 and PD98059 were from Cell Signaling Technology Inc.

Cell culture medium components were purchased from Thermo Fisher Scientific (Waltham, MA, USA) unless otherwise indicated. The polyclonal antibodies used in the study were: (i) STAT3 (cat. no. 4904), phospho-STAT3 (pSTAT; cat. no. 3l9145), Smad2/3 (cat. no. 3102), phospho-Smad2/3 (pSmad2/3; cat. no. 8828), Smad4 (cat. no. 38454) and Snail (cat. no. 3879) from Cell Signaling Technology, Inc. (Danvers, MA, USA); (ii) E-cadherin (E-cad; cat. no. 610182) from BD Biosciences (San Jose, CA, USA); (iii) N-cad (cat. no. sc-59987) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); (iv) α-SMA (cat. no. ab5694) and fibronectin (cat. no. ab2413) from Abcam (Cambridge, UK); (v) glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cat. no. A300-641A) and horseradish peroxidase (HRP)-conjugated secondary antibodies from Bethyl Laboratories, Inc. (Montgomery, TX, USA). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Day 26 differentiated neuroepithelial cells were evaluated via immunocytochemical staining of Pax6 (Biolegend, San Diego, CA; 901301, RRID:AB_2565003), NCad (BD Biosciences, San Jose, California; 610920, RRID:AB_2077527), ZO-1 (InVitrogen, 33–9100, RRID:AB_2533147), aPKCζ (Santa Cruz, Dallas, Texas; sc-216, RRID:AB_2300359), Pericentrin (Abcam, ab28144, RRID:AB_2160664), and pHH3 (Santa Cruz, sc-12927, RRID:AB_2233069). Day 45 immature neurons were stained for Tuj1 (Abcam, ab78078, RRID:AB_2256751) and NeuN (Abcam, ab177487, RRID:AB_2532109). The secondary antibodies were donkey anti-rabbit IgG conjugated with Alexa-488 (Jackson, Bar Harbor, Maine; 711545152, RRID:AB_2313584), donkey anti-mouse IgG conjugated with Cy3 (Jackson, 715165150, RRID:AB_2340813), and donkey anti-goat IgG conjugated with Cy5 (Jackson, 705175147, RRID:AB_2340415). Quantification of pHH3 was performed using ImageJ (RRID:SCR_003070) and Adobe Photoshop (RRID:SCR_014199), while all statistical evaluations were performed with GraphPad Prism v7.04 (RRID:SCR_002798).

Immunofluorescent staining was performed, as previously described (Veeraraghavan et al., 2015 (link); Veeraraghavan et al., 2016 (link); Veeraraghavan and Gourdie, 2016 (link)), on 5 µm cryosections of tissue, and monolayers of cells fixed with paraformaldehyde (2%; 5 min at room temperature). Samples were labeled with our novel rabbit polyclonal antibodies against either Na_V1.5 (Epitope: ₁₉₉₆HSEDLADFPPSPDRDRESIV₂₀₁₆) or β1 (Epitope: ₄₄KRRSETTAETFTEWTFR₆₀). Validation results for these antibodies are presented in Figure 1—figure supplement 1. In some cases, samples were co-labeled with a mouse monoclonal antibody against either connexin43 (Cx43; Millipore MAB3067, 1:250) or N-cadherin (N-cad; BD Biosciences 610920, 1:100). For confocal microscopy, samples were then labeled with goat anti-rabbit AlexaFluor 568 (1:4000; ThermoFisher Scientific, Grand Island, NY) and goat anti-mouse AlexaFluor 633 (1:4000; ThermoFisher Scientific, Grand Island, NY) secondary antibodies. For super-resolution STochastic Optical Reconstruction Microscopy (STORM), samples were labeled with goat anti-rabbit Alexa 647 (1:4000) and donkey anti-mouse Cy3b (1:100) secondary antibodies (ThermoFisher Scientific, Grand Island, NY) and stored in Scale U2 buffer (Hama et al., 2011 (link)) for 48 hr at 4°C.

Immunofluorescence antibodies [target, dilution, (species, company)]. Primary antibodies: FHF2 (ref. 27 (link)) 1:500 (Epitope GGKSMSHNEST, Rabbit), Na_V1.5 1:50 (mouse, Alomone, ASC-005), Sarcomeric Actinin 1:100 (mouse, Sigma, a7811), N-cad 1:100 (mouse, BD Biosciences, 610921). Mounting medium with DAPI (Vectashield, H-1200).

For immunoblotting cells were grown on Matrigel and treated as represented in the image. In the case of GLT combination treatments, cells were pretreated with GLT and then with TGFβ1. Post 24 h of treatment, cells were trypsinized and washed with cold PBS to dissolve Matrigel. The cells were lysed in protease and phosphatase inhibitor (Thermo-Fisher) containing RIPA (Boston Bioproducts) buffer. The protein content of the lysate were quantified using BCA assay (Thermo Fisher Scientific) and western blot was performed with equal protein. Membranes were blocked for 1 h at room temperature (5% nonfat milk) followed by overnight incubation with primary antibody. Following primary antibodies were used – p Smad-2 (#18338), Smad-2 (#5339), SNAI1 (#3879), Cyclin D1 (#55506), (Cell signaling Technology), Fibronectin (#610077), N-Cad (#610921)(BD Biosciences), GAPDH (#G9295), and α tubulin (#T5201) (Sigma), Acidic and basic Cytokeratin AE1/AE3 (#MAB3412) (Millipore) and EMA (#GA62961) (Dako). Secondary antibodies (from sigma) were used at a concentration of 1:10,000. Equal loading was verified by immunoblotting with GAPDH or αTubulin.