Fig. 4: Genetic validation of cell surface membrane U5 snRNP200 expression and determinants of U5 snRNP200 cell surface membrane localization.
a, Schema of the vector engineered for knock-in of the sequence for N-terminal HaloTag into the SNRNP200 locus in the human AML cell line K562. UTR, untranslated region. HA, homology arm. b, PCR amplification of the HaloTag sequence for verification of expression in HaloTag–U5 snRNP200-expressing K526 cells. Representative of three independent experiments. c, Western blot for HaloTag (two exposure times are shown and denoted as light and heavy) and U5 snRNP200 in subcellular fractions of K562 cell clones expressing HaloTagged endogenous U5 snRNP200. Loading controls for cell fractions include tubulin (cytoplasmic), Na+/K+ ATPase pump (membrane) and specificity protein 1 (SP1; soluble, nuclear). WT, wild type. d, Representative flow cytometry histograms of MFI values (left) for cell-impermeable (top) and cell-permeable (bottom) fluorescent HaloTag ligands in K562 cells from b and quantification of signal (right; mean value is shown and each dot represents a data point from an independent experiment). AF660, Alexa Fluor 660. TMR, tetramethylrhodamine.
