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. 2019 Feb 19;93(5):e01714-18. doi: 10.1128/JVI.01714-18

FIG 5.

FIG 5

Generation and validation of MDBK and SK6 DNAJC14-KO rescue cell lines expressing GST-Jiv90(WT) or GST-Jiv90(W39A). (A) Scheme of the lentiviral expression constructs pWPI-GST-Jiv90(WT) and pWPI-GST-Jiv90(W39A). LTR, long terminal repeat. (B) Schematic of the experimental approach used for the generation of the MDBK and SK6 DNAJC14-KO rescue cells. (C) Analysis of GST-Jiv90 expression in MDBK DNAJC14-KO rescue cells by IF. DNAJC14-KO cells and DNAJC14-KO rescue cells expressing GST-Jiv90(WT) or GST-Jiv90(W39A) were stained with anti-DNAJC14 (recognizing epitope SARYCAECNR in the Jiv90 domain) or anti-GST-specific antibodies. Cell nuclei were counterstained with DAPI. (D) Western blot analysis of GST-Jiv90 expression in MDBK DNAJC14-KO rescue cells. Protein lysates obtained from naive MDBK and MDBK DNAJC14-KO cells and MDBK DNAJC14-KO rescue cells expressing either GST-Jiv90(WT) or GST-Jiv90(W39A) were separated by SDS-PAGE and analyzed with anti-GST (top) or anti-DNAJC14 (bottom). The positions of DNAJC14 GST-Jiv90(WT) and GST-Jiv90(W39A) are indicated on the right, and the protein marker positions are given on the left. (E) Analysis of GST-Jiv90 expression in SK6 DNAJC14-KO rescue cells by anti-GST- and anti-DNAJC14-specific IF. (F) Western blot analysis of GST-Jiv90 expression in SK6 DNAJC14-KO rescue cells expressing either GST-Jiv90(WT) or GST-Jiv90(W39A). Protein lysates obtained from naive SK6, SK6 DNAJC14-KO cells, and SK6 DNAJC14-KO rescue cells expressing either GST-Jiv90(WT) or GST-Jiv90(W39A) were separated by SDS-PAGE and analyzed with anti-GST (top) or anti-DNAJC14 (bottom). The positions of DNAJC14 GST-Jiv90(WT) and GST-Jiv90(W39A) are indicated on the right, and the protein marker positions are given on the left.