Blocking PIKFYVE with apilimod does impacts TCR internalization but not Zap70 activation. (A) A flow cytometry–based internalization assay was performed to monitor the extracellular and intracellular levels of the TCR/CD3 complex after Th17 polarization ± apilimod treatment (N = 4). (B) Naive murine CD4 T cells were isolated by negative selection and treated with apilimod (100 nM) or vehicle for 1 h and activated under Th17 polarization conditions. Flow cytometry was utilized to measure the abundance of pZAP70(Y319), expressed by mean fluorescence intensity (MFI), (N = 3 per datapoint).