Patient mutant CD3ζ fails to rescue TCR expression in retrovirally-transduced CD3ζ-deficient MA5.8 cells. (A) MA5.8 cells were transduced with individual retroviral vectors containing one of the following human CD3ζ cDNAs: hsCD3ζWT (wild-type CD3ζ transcript variant 2), hsCD3ζCm (patient 411insC mutant CD3ζ transcript variant 2), hlCD3ζWT (wild-type CD3ζ transcript variant 1), or hlCD3ζCm (patient mutant CD3ζ transcript variant 1). Cells were also transduced with pMiG vector alone. Depicted are 1-color histograms of transduced MA5.8 cells analyzed for expression of eGFP indicator protein, surface TCRβ, or surface CD3ε by flow cytometry. (B) MA5.8 cells retrovirally transduced with the indicated cDNAs were lysed in digitonin lysis buffer, resolved by SDS-PAGE, and immunoblotted with anti-CD3ζ Ab (top panel) and anti-CD3ε Ab (middle panel). The membrane was also probed with anti-Calnexin Ab (bottom panel) to assess gel loading.