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. 2016 Sep 1;7:12702. doi: 10.1038/ncomms12702

Figure 2. ORP4L facilitates assembly of a signalling complex in T-ALL cells.

Figure 2

(a) SDS–PAGE gel silver-stained for control IgG and anti-ORP4L immunoprecipitated proteins from Jurkat T-cells. The lane marked IB displays control IgG and anti-ORP4L immunoprecipitated proteins analyzed by western blot with anti-PLCβ3, Gαq/11 and CD3ɛ antibodies simultaneously. (b) Co-immunoprecipitation analysis of ORP4L binding proteins in Jurkat T-cells. Cells were incubated with 10 μg ml−1 of anti-CD3 for the indicated times before lysates. (c) Co-immunoprecipitation analysis of Gαq/11 binding to CD3ɛ and PLCβ3 in control and ORP4L knockdown cells. Cells were infected with shNT or shORP4L lentivirus for 72 h, and then incubated with or without 10 μg ml−1 of anti-CD3 for 5 min before lysates. (d) Co-immunoprecipitation analysis of Gαq/11 binding to CD3ɛ and PLCβ3 in Jurkat T-cell lysates before immunodepletion with ORP4L antibody. (e) Co-immunoprecipitation analysis Gαq/11 binding to CD3ɛ and PLCβ3 in normal T-cells. Cells were incubated with or without 10 μg ml−1 of anti-CD3 for 10 min before lysates. (f) Confocal immunofluorescence microscopy analysis of CD3ɛ (green), ORP4L (red) and pan-Cadherin (blue) localization in shNT and shORP4L transduced Jurkat T-cells. Scale bars, 10 μm. (g) Confocal immunofluorescence microscopy analysis of Gαq/11 (green), ORP4L (red) and pan-Cadherin (blue) localization in shNT and shORP4L transduced Jurkat T-cells. Scale bars, 10 μm. (h) Confocal immunofluorescence microscopy analysis of PLCβ3 (green), ORP4L (red) and pan-Cadherin (blue) localization in shNT and shORP4L transduced Jurkat T-cells. Scale bars, 10 μm. (i–k) Confocal microscopy analysis of CD3ɛ (blue), Gαq/11 (green) and p–PLCβ3 (red) localization in shNT and shORP4L transduced Jurkat T-cells (i), Molt-4 cell (j) and primary T-ALL cells (k). Scale bars, 10 μm. For the confocal immunofluorescence above, cells were infected with shNT or shORP4L lentivirus for 72 h and then stimulated before staining for 5 min with or without 10 μg ml−1 anti-CD3.