Clone 36

For T cell stimulations anti-CD3, clone 145-2C11 (BD 553057) and anti-CD28, clone 37.51 (BD 553295) were used. For immunoprecipitations (IP) and immunoblotting (IB): Mouse anti-Dlg1 (BD 610875), Rabbit anti-p38, clone C20 (Santa Cruz Biotechnology sc-535), Mouse anti-Lck, clone 3A5 (Santa Cruz Biotechnology sc-433), Mouse anti-ZAP70 (BD 610239) Mouse anti-WASp, clone B-9 (Santa Cruz Biotechnology, sc-13139), Rabbit anti-phospho-p38 (T180/Y182), clone D3F9 (Cell Signaling 4511), Donkey Anti-Mouse IgG-HRP (Jackson ImmunoResearch 715-035-150), and Donkey-Anti-Rabbit IgG-HRP (Santa Cruz Biotechnology sc-2305) were used. For flow cytometry, Rat anti-CD8b-PE, clone H35-17.2 (BD 550798), Alexa Fluor647 Mouse anti-p38 MAPK (pT180/pY182), clone 36 (BD 612595), Rat anti-IFNγ-APC, clone XMG1.2 (BD 554413), Rat anti-IL-2-APC, clone JES6-5H4 (BD 554429), Rat anti-TNFα-APC (BD 554420), Rat anti-CD107a-APC, clone 1D4B (BD 560646), Alexa Fluor647 Phalloidin (Molecular Probes, Invitrogen A22287), Mouse monoclonal anti-WASP 26E6 [22 (link)] and Alexa Fluor647 AffiniPure F(ab)2 Donkey Anti-Mouse IgG (Jackson ImmunoResearch 715-606-150) were used. For inhibitors studies Insolution SB203580 (Calbiochem 559398), cytochalasin D (Sigma C8273) and DMSO (Sigma D2650) were used. For antigen experiments OVA_257-264 peptide from AnaSpec (#60193) was used.

Cells grown on coverslips were fixed in absolute ethanol for 20 min. Samples were blocked in incubation buffer (5% normal goat serum and 0.1% BSA in PBS) for 1 h at room temperature. Primary antibody was diluted in 0.1% BSA/PBS and incubated 1 h at room temperature. The following primary antibodies and dilutions were used: mouse anti-E-cadherin (1:1000; clone 36, BD Biosciences, Stockholm, Sweden) and vimentin (1:1000; clone VI-10, Abcam, Cambridge, UK). Cells were washed five times in PBS/BSA and further incubated with secondary antibodies (1:1000 dilution) for 30 min and mounted in vectashield mounting media supplemented with DAPI (Vector Laboratories Ltd, Peterborough, UK). Immunoflouresent staining was assessed using a Nikon Eclipse microscope equipped for immunofluorescence.

Heads dissected from E12.5, E13.5 and E14.5 mice were fixed in 4% paraformaldehyde and processed for histological examination using standard protocols. For immunofluorescence analyses, sections were treated with 10 mm citrate buffer at 96°C for 10 min for antigen retrieval. Sections were incubated overnight at 4°C with antibodies against E-cadherin (1:200; Clone-36, BD Bioscience), keratin 17 (1/1000), keratin 14 (1/200, clone MS-115-P1, Neomarkers) nectin-1, (1:200; Santa Cruz SC-28639), nectin-4 (1:100; HPA010775 Sigma), plakoglobin (1/200; clone 15F11 Sigma), p63 (1:1000; Santa Cruz 4A4) and Bcl11b (1/500 Ctip2, clone 25B6, Abcam). In situ hybridization was performed as described previously with detection using BM Purple (Roche) [8 (link)]. Sections were counterstained and visualized using a Leica DMRB microscope. Littermates were used as controls wherever possible, in order to correctly stage the mutant (Tgfb3^-/-;p63^+/- or Krt5-tTA;pTRE-ΔNp63α bi-transgenic embryos), and to provide control tissue. In all cases, the histological analyses were performed on at least five mutant embryos from all gestational ages (two for the neonatal Krt5-tTA;pTRE-ΔNp63α bi-transgenic embryos due to ethical considerations). Immunofluorescence and in situ hybridization assays were performed in duplicate.