FIGURE

Fig. 5

ID

ZDB-FIG-200306-53

Publication

Lee et al., 2020 - REEP5 depletion causes sarco-endoplasmic reticulum vacuolization and cardiac functional defects

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Fig. 5

The C-terminal cytosolic domain of REEP5 is required for stabilizing SR/ER morphology.

a Schematic diagrams of REEP5 constructs used for REEP5 dimerization study shown in Fig. 5b–d. b Immunoblot analysis of endogenous REEP5 monomer (~17 kDa) and REEP5 dimer (~34 kDa), and exogenous REEP5 monomer (~22 kDa) and exogenous REEP5 dimer (~44 kDa) in HEK293 transfected with REEP5-V5-6xHis construct. c Immunoblots of REEP5 dimer dissociation in response to increasing DTT concentration. Diamonds to the left indicate the detection of endogenous (orange) and exogenous (blue) REEP5 monomers and dimers. d Co-immunoprecipitation assays in HEK293 cells. REEP5-V5 and CnVA-REEP5 were transfected into cells and precipitated with V5 or flag antibody. Left, control immunoblot experiments. Right panel, CnVA-REEP5 was immunoprecipitated via anti-V5 antibody in REEP5-V5-6xHis transfected HEK293 cells. e Immunoblot analysis of C₂C₁₂ cells transfected with REEP5 truncation mutants shows depletion of REEP5 dimers in the absence of the carboxyl terminal domain. f Confocal imaging analysis of EYFP (green) and DAPI (blue) staining shows truncation of the carboxyl terminal domain of REEP5 (Δ114–189) causes ER luminal vacuolization in transfected C₂C₁₂ myoblasts. Scale, 10 μm. Right, image intensity analysis along axis. g Schematic diagram of truncated REEP5 mutant constructs fused with GFP. Amino acid sequences are shown. h Live-cell confocal imaging of C₂C₁₂ myoblasts expressing the carboxyl-terminus truncated mutant of REEP5 (Δ114–189) and recombinant mCherry-fused mitochondrial targeting signal (mCherry-mito) or luminal ER marker (mCherry-KDEL). Scale, 10 μm. i Live-cell confocal imaging of C₂C₁₂ myoblasts expressing four truncated REEP5 mutants co-expressed with mCherry-KDEL suggests the importance of the C-terminal cytosolic domain of REEP5 in stabilizing ER membrane curvatures. Scale, 10 μm. Source data containing original uncropped immunoblots are provided as a Source Data file. All images shown are representative of approximately 40–50 total images captured per condition, n = 3 independent biological replicates.

Expression Data

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Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

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