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TECPR2 directly binds to Rab5 via its C-terminal TECPR repeats. A Domain architecture of TECPR2. Green and red dots indicate the HSAN9-associated variants and TECPR2 founder variants, respectively. B, C Yeast two-hybrid assay of TECPR2 with GTP-locked mutants of selected Rab proteins (B) and with Rab5 (WT) and mutants (C). Co-transformants were spotted on -Leu/-Trp and -Leu/-Trp/-His to confirm viability and detect interactions, respectively. D HEK293T cell lysates expressing indicated GFP-tagged proteins were immunoprecipitated with anti-GFP and immunoblotted (IB) with the indicated antibodies. The anti-GFP nanobody was visualized using Ponceau S staining. Densitometric analysis of TECPR2, normalized to the input and direct IP, is shown. E HEK293T cell lysates were immunoprecipitated with anti-Rab5 or mouse IgG (used as a control) and IB with indicated antibodies. F Yeast two-hybrid assay of TECPR2 (WT) and its domain-deletion mutants with Rab5 (Q79L). Co-transformants were spotted on -Leu/-Trp, -Leu/-Trp/-His, and -Leu/-Trp/-His/-Ade media to confirm viability and interactions, respectively. G MBP and MBP-TECPR2_TECPR proteins were used to pull down the indicated GFP-tagged Rab5 (WT) or mutant proteins expressed in HEK293T cells. The GFP-tagged proteins were detected by IB and MBP proteins using Ponceau S staining. Densitometric analysis of GFP-Rab5 (WT) and mutant proteins, normalized to the input and direct pulldown, is shown. SE = Short Exposure; LE = Long Exposure. H GST and GST-Rab5 (WT) and mutants were used to pull down the MBP-TECPR2_TECPR protein. The MBP-tagged proteins were identified by IB and GST proteins using Ponceau S staining. Densitometric analysis of TECPR2_TECPR protein, normalized to the input and direct pulldown, is shown. I HEK293T cell lysates co-expressing indicated GFP- and FLAG-tagged proteins were immunoprecipitated with anti-GFP and IB with the indicated antibodies. The anti-GFP nanobody was visualized using Ponceau S staining. Densitometric analysis of TECPR2 (WT) and mutants, normalized to the input and direct IP, is shown. J Yeast two-hybrid assay of TECPR2 HSAN9-associated variants with Rab5 (Q79L). Co-transformants were spotted on -Leu/-Trp and Leu/-Trp/-His media to confirm viability and detect interactions, respectively. K HEK293T cell lysates co-expressing indicated GFP- and FLAG-tagged proteins were immunoprecipitated with anti-GFP and IB with the indicated antibodies. The anti-GFP nanobody was visualized using Ponceau S staining. Densitometric analysis of TECPR2 (WT) and mutants, normalized to input and direct IP, is shown. Source data are provided as a Source Data file.
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