Journal:

Article Title: Two adaptor proteins differentially modulate the phosphorylation and biophysics of Kv1.3 ion channel by Src kinase

doi: 10.1074/jbc.M108898200

Figure Lengend Snippet: Human embryonic kidney (HEK 293) cells were transiently transfected with Kv1.3 cDNA plus or minus v-Src kinase and plus or minus adaptor protein (grb10 or n-shc). A, (top panel), Kv1.3 protein was immunoprecipitated from Triton X-100-soluable cell lysates with α-Kv1.3 antiserum (IP:αKv1.3), separated by SDS-PAGE, electrotransferred to nitrocellulose, and probed with α-phosphotyrosine antibody (IB:αPY) that recognizes phosphorylated tyrosine residues. Upper arrow indicates Mr of Kv1.3 and lower arrow indicates the heavy chain of IgG (IgG). The box defines the area that was integrated (55-72 kDa) to generate the quantitative immunodensity data in B. A, (lower panel), Cell lysates used in the above immunoprecipitation experiment were probed with α-Kv1.3 antiserum to insure equal protein expression of the channel under varying transfection conditions. B, Histogram plot of the tyrosine phosphorylation of Kv1.3 (Kv1.3), as measured by quantitative immunodensitometry, indicates an increase in phosphorylation in the presence of v-src (+v-src) or v-src plus n-shc (+v-src + n-shc). This increase in v-src induced tyrosine phosphorylation of Kv1.3 is inhibited in the presence of grb10 (+ v-src + grb10). Pixel counts for each band were normalized to the value of the Kv1.3-only band within single gels for graphical representation. Non-normalized values from each transfection condition were compared statistically with one-way randomized design ANOVA, α = 0.05. * = significantly different from control (Kv1.3) by snk follow-up test.

Article Snippet: Neuronal Shc (n-Shc), Sck, and n-Shc mutant cDNA (Y to F point mutations at tyr 220 , tyr 221 , tyr 304 ) were generous gifts from Dr. T. Nakamura (Sumitomo Electric Industries, Yokohama, Japan) and were expressed in the vector pCMV1 { 32 }.

Techniques: Transfection, Immunoprecipitation, SDS Page, Expressing, Phospho-proteomics, Control

Journal:

Article Title: Two adaptor proteins differentially modulate the phosphorylation and biophysics of Kv1.3 ion channel by Src kinase

doi: 10.1074/jbc.M108898200

Figure Lengend Snippet: Human embryonic kidney (HEK 293) cells were transiently transfected with Kv1.3 cDNA +/- v-src +/- grb10 or n-shc, as in Fig. 3. A-B, Representative cell-attached patch-clamp recordings from HEK 293 cells that were held at -90 mV (Vh) and stepped to +40 mV (Vc) for 1000 msec. Three different HEK 293 cells are shown in each, demonstrating Kv1.3 alone (Kv1.3), Kv1.3 + v-src (+ v-src) and Kv1.3 + v-src + adaptor protein (+ v-src + grb10) or (+v-src + n-shc) transfection conditions. C-D, Same transfection and recording conditions as in A-B, respectively, but traces are normalized to peak current value to visualize rate of inactivation (τinact). E-F, Same transfection conditions as in A-B, respectively, but traces are normalized to peak tail current to visualize rate of deactivation (τdeact). τinact and τdeact were determined by fits to bi-exponential or single exponential functions, respectively, as described in Experimental Procedures. A-F, Values from transfection conditions were compared statistically by one-way completely randomized design ANOVA, snk follow-up test, α = 0.05. * = significantly different from control (Kv1.3). ** = significantly different from Kv1.3 + v-src co-transfection.

Article Snippet: Neuronal Shc (n-Shc), Sck, and n-Shc mutant cDNA (Y to F point mutations at tyr 220 , tyr 221 , tyr 304 ) were generous gifts from Dr. T. Nakamura (Sumitomo Electric Industries, Yokohama, Japan) and were expressed in the vector pCMV1 { 32 }.

Techniques: Transfection, Patch Clamp, Control, Cotransfection

Journal: Science Advances

Article Title: Identification of the Wnt signal peptide that directs secretion on extracellular vesicles

doi: 10.1126/sciadv.ado5914

Figure Lengend Snippet: ( A ) SP is not required for EVs-Wnt7a secretion in transfected HEK293T cells. ( B ) Mutant Wnt7a_S206A, lacking the palmitoylation site, is secreted on EVs in transfected HEK293T cells. ( C ) Drug inhibition of PORCN does not affect secretion of Wnt7a on EVs. ( D ) Knockdown of WLS in siRNA partially affects secretion of Wnt7a on EVs but does abolish secretion of non-EV Wnt7a. ( E ) BirA constructs for BioID analysis. ( F ) Heatmap displaying fold change (log 2 scale) of enriched proteins in mass spectrometry (ESP_BirA:BirA and Wnt7a_BirA:BirA). Shown are enrichment of >50% [log 2 (FC) > 0.5849] on EBP and a positive enrichment [log 2 (FC) > 0] on Wnt7a. ( G ) Wnt7a: COPA PLA (red) performed in murine primary myotubes either expressing Wnt7a-BirA or BirA. GM310 in green and 4′,6-diamidino-2-phenylindole (DAPI) in blue. Scale bars, 10 μm. ( H ) Wnt7a:COPA PLA (orange) performed in RPTEC- hTERT1 cells. Wheat Germ Agglutinin (WGA) in green and DAPI in blue. CTR-neg is control without Wnt7a antibody. Scale bars, 10 μm. ( I ) Wnt7a is secreted on EVs derived from RPTEC-hTERT1 cells. HEK293T cells lacking Wnt7a were used as a negative control. ( J ) Wnt7a:COPA PLA (red) performed in HEK293T cells expressing Wnt7a-FL, Wnt7a_ΔEBP*GSGS, or Wnt7a_ΔSP. GM310 in green and DAPI in blue. Scale bars, 10 μm. CTR-neg is control without Wnt7a antibody. ( K ) Wnt7a-HA interacts with COPA and COPB2. HEK293T cells overexpressing Wnt7a-HA were immunoprecipitated with COPB2 antibody or ( L ) immunoprecipitated with HA antibody. ( M ) Immunoblot EVs secretion analysis of Wnt7a after siRNA knockdown of COPA and COPB2 shows disruption of Wnt7a-EV secretion in transfected HEK293T cells. n = 3 biological replicates. IgG, immunoglobulin G; WGA, Wheat Germ Agglutinin.

Article Snippet: The DNA sequence encoding COPB2 1–304 was custom-synthesized with codon optimization for expression in Escherichia coli (GenScript) and cloned into pET28-Sumo3 vector (EMBL, Heidelberg) to express it with an N-terminal cleavable 6xHis-Sumo3 tag.

Techniques: Transfection, Mutagenesis, Inhibition, Knockdown, Construct, Mass Spectrometry, Expressing, Control, Derivative Assay, Negative Control, Immunoprecipitation, Western Blot, Disruption

Journal: Science Advances

Article Title: Identification of the Wnt signal peptide that directs secretion on extracellular vesicles

doi: 10.1126/sciadv.ado5914

Figure Lengend Snippet: ( A ) Predicted Wnt7a structure in AlphaFold (blue) with the EBP region highlighted in orange and the three positively charged motifs (red) within the EBP. Note that EBP is a solvent-exposed region. ( B ) Wnt7a-ESP*Scramble mutant maintains the dilysine motif and exhibits no impairment in EV secretion. ( C ) ITC measurements for COPB2 1–304 binding to potential dilysine/arginine motifs within the EBP. WT COPB2 1–304 binds to the LKIKKP subregion. ( D to F ) views of the KxKx motif of Wnt7a bound to COPB2 1–304 . (D) Top view of the WD-repeat domain of COPB 1–304 (green) with the LKIKKP peptide (orange) in ribbon representation. (E) Close-up view of the LKIKKP peptide with a difference electron density map calculated by omitting the peptide and contoured at 3σ (blue mesh). COPB2 surface is colored by electrostatic potential ranging from −5 kT/e (red) to 5 kT/e (blue). (F) Lateral view of the binding motif with hydrogen bonds and distances. ( G ) Structure-based point mutations confirm the molecular recognition of the KxKx motif in ITC assays. ( H ) Double lysine mutation of K253 and K255 by alanine disrupts Wnt7a-EV secretion. ( I ) Replacement of Wnt7a-EBP by either Wnt10a-EBP or Wnt16 EBP containing KR and RR (right). Replacement with Wnt10a-EBP or Wnt16 EBP rescues Wnt7a-EV secretion. ( J ) EV secretion analysis of Wnt10b after EBP removal or double arginine mutation within its EBP (right). Double arginine mutation disrupts secretion of Wnt10b on EVs to the same extent as removal of the entire Wnt10b EBP sequence. ( K ) Secretion analysis of Wnt3a after EBP removal or mutation of the entire positively charged motifs RPR, KHR, and KH within its EBP (right). Only concomitant mutation of RPR and KHR motifs disrupts secretion of Wnt3a on EVS to the same extent as removal of the entire Wnt3a EBP sequence. n = 3 biologic replicates.

Article Snippet: The DNA sequence encoding COPB2 1–304 was custom-synthesized with codon optimization for expression in Escherichia coli (GenScript) and cloned into pET28-Sumo3 vector (EMBL, Heidelberg) to express it with an N-terminal cleavable 6xHis-Sumo3 tag.

Techniques: Solvent, Mutagenesis, Binding Assay, Sequencing

Journal: Science Advances

Article Title: Identification of the Wnt signal peptide that directs secretion on extracellular vesicles

doi: 10.1126/sciadv.ado5914

Figure Lengend Snippet: ( A ) Knockdown of COPA and COPB2 using siRNA disrupts Wnt7a-EV secretion in Wnt7a stably expressing primary myoblasts. ( B ) Knockdown of COPA and COPB2 decreases hypertrophy in Wnt7a-transfected myotubes. Conversely, knockdown did not affect hypertrophy in myotubes not expressing Wnt7a. ( C ) Representative images after simultaneously siRNA knockdown of COPA and COPB2 in myotubes. Scale bars, 50 μm. ( D ) Schematic representation of in vivo workflow. ( E ) Myofiber caliber distribution comparing TA electroporated with Wnt7a (green) versus Wnt7a_ΔEBP*GSGS (gray). ( F ) Average minimal fiber feret comparing TA electroporated with Wnt7a (green) versus Wnt7a_ΔEBP*GSGS (gray). ( G ) Quantification of fiber number comparing TA electroporated with Wnt7a (green) versus Wnt7a_ΔEBP*GSGS (gray). ( H ) Quantification of muscle area comparing TA electroporated with Wnt7a (green) versus Wnt7a_ΔEBP*GSGS (gray). ( I ) Section of TA muscles showing reduced myofiber caliber an increased number of myofibers after electroporation of Wnt7a_ΔEBP*GSGS into TA muscle of mdx mice. Scale bars, 100 μm. TA, tibialis anterior. In vitro experiments are representative of three independent biological replicates performed in murine primary myoblasts. In vivo experiments are representative n = 6 mice, means ± SEM. P value was determined by two-sided Student’s t test (* P < 0.05 and ** P < 0.005).

Article Snippet: The DNA sequence encoding COPB2 1–304 was custom-synthesized with codon optimization for expression in Escherichia coli (GenScript) and cloned into pET28-Sumo3 vector (EMBL, Heidelberg) to express it with an N-terminal cleavable 6xHis-Sumo3 tag.

Techniques: Knockdown, Stable Transfection, Expressing, Transfection, In Vivo, Muscles, Electroporation, In Vitro