Journal: Journal of cell science

Article Title: Tubular microdomains of Rab7-positive endosomes retrieve TrkA, a mechanism disrupted in Charcot-Marie-Tooth disease 2B.

doi: 10.1242/jcs.258559

Figure Lengend Snippet: Fig. 6. WASH1 is involved in late receptor tubulations. (A) Anti-GFP-conjugated beads were used to immunoprecipitate (IP) GFP, endophilinA1–GFP (A1–GFP), endophilinA2– GFP (A2–GFP) or endophilinA3–GFP (A3–GFP) with TrkA–RFP from co-transfected HEK293 cells (input on the left, IP on the right) in the presence or absence (NF) of 100 ng/ml NGF. (B) Anti-RFP-conjugated beads (or IgG control beads) were used to pull down EGFR–RFP with endophilinA1–GFP, endophilinA2–GFP or endophilinA3–GFP from co-transfected HEK293 cells in the presence or absence (NF) of 100 ng/ ml EGF (input on the left, IP on the right). Data in A and B are representative of three experiments. (C) Immunostaining of MEFs showing WASH1 on the rim of late Rab7 vacuoles when stimulated with NGF. Scale bar: 20 µm. (D) Immunostaining of MEFs showing WASH1 not localizing to late Rab7 vacuoles when stimulated with EGF. Scale bar: 20 µm. In C and D, boxes indicate regions shown as magnified images. Lines indicate transects shown in plot profiles. Nuclei are stained with DAPI. Images are representative of three experiments. (E) Colocalization of stained WASH1 and Rab7 increases in DRGs upon stimulation with NGF. Boxes indicate regions shown in magnified images on the right. Arrowheads indicate colocalization. Quantification shows mean±s.e.m. Pearson’s correlation coefficient. n=10–15 images per condition, the experiment was performed three times. **P=0.0046 (two-tailed, unpaired t-test). Scale bar: 10 µm. (F) EndophilinAs co- immunoprecipitate with WASH1 in lysates from HEK293 cells co-transfected with GFP-tagged endophilinAs and WASH1–mCherry. Anti-GFP- conjugated beads (or control IgG beads) were used for the IP [input and output (protein levels after incubation with conjugated beads) on the left, IP on the right]. Blots shown are representative of three experiments.

Article Snippet: Immunofluorescence was performed using the following antibodies: TrkA polyclonal rabbit antibody (Millipore, 06-574), EGFR (A10) mouse monoclonal antibody (Santa Cruz, sc-373746), p-TrkA Y794 polyclonal rabbit antibody (Millipore, ABN1383), pEGFR Y1068 monoclonal rabbit antibody (Cell Signaling Technologies, 3777), βIIItubulin mouse monoclonal antibody (Abcam, ab78078), WASH1 polyclonal rabbit antibody (Sigma, SAB4200372), endophilinA1 mouse monoclonal antibody (Santa Cruz, sc-374279), endophilinA2 mouse monoclonal antibody (Santa Cruz, sc-365704), endophilinA2 rabbit polyclonal antibody (Proteintech, 27014-1-AP), endophilinA3 mouse monoclonal antibody (Santa Cruz, sc-376592), Rab7 mouse monoclonal antibody (Cell Signaling Technologies, 95746), Rab7 polyclonal rabbit antibody (Synaptic Systems, 320 003).

Techniques: Transfection, Control, Immunostaining, Staining, Two Tailed Test, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli : Dissecting and Mitigating Redox Heterogeneity

doi: 10.3390/ijms232314740

Figure Lengend Snippet: SDS-PAGE analysis of seven candidate Fc fusion proteins produced solubly with the help of the CyDisCo system in the cytoplasm of E. coli ( A ) SDS-PAGE gel image of T: total cell lysate and S: soluble cell lysate under reducing conditions. ( B ) SDS-PAGE gel image of Protein G-based purified POIs and purified wild type IgG 1 Fc region in three different concentrations under reducing conditions. ( C ) SDS-PAGE gel image of Protein G-based purified POIs under non-reducing and N-Ethyl maleimide (NEM)-treated conditions showing redox heterogeneity. M: marker, 1: Trebananib, 2: Leptin–Fc, 3: hGH–Fc, 4: Angiotensin–Fc, 5: Substance P–Fc, 6: Gastrin–Fc, 7: Katacalcin–Fc, S1: IgG 1 Fc region (0.19 µg), S2: IgG 1 Fc region (0.38 µg), S3: IgG 1 Fc region (0.56 µg).

Article Snippet: Genes for Trebananib fusion partner and Leptin with BamHI-EcoRI flanking sites were synthesized via codon optimization (GenScript Biotech Corp.) for E. coli expression.

Techniques: SDS Page, Produced, Purification, Marker

Journal: International Journal of Molecular Sciences

Article Title: Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli : Dissecting and Mitigating Redox Heterogeneity

doi: 10.3390/ijms232314740

Figure Lengend Snippet: Yields of wild type Fc fusion proteins purified from 3 mL cultures (24 DWP).

Article Snippet: Genes for Trebananib fusion partner and Leptin with BamHI-EcoRI flanking sites were synthesized via codon optimization (GenScript Biotech Corp.) for E. coli expression.

Techniques: Purification

Journal: International Journal of Molecular Sciences

Article Title: Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli : Dissecting and Mitigating Redox Heterogeneity

doi: 10.3390/ijms232314740

Figure Lengend Snippet: ( A ) SDS-PAGE analysis of wild type and mutant IgG 1 Fc region under non-reducing and NEM-treated conditions showing that the mutating the cysteines in the C H 3 domain to alanine results in the production of the IgG 1 Fc region in a single homogeneous redox state. M: protein marker, 1: IgG 1 Fc region wild type, Lane 2: IgG 1 Fc region (C250A, C308A). ( B ) SDS-PAGE gel image of Protein G-based purified POIs with a mutant IgG 1 Fc region (C250A, C308A) and purified wild type IgG 1 Fc region in three different concentrations under reducing conditions. ( C ) SDS-PAGE gel image of Protein G-based purified POIs with a mutant IgG 1 Fc region (C250A, C308A) under non-reducing and NEM-treated conditions showing that the fusion proteins are produced in a single homogeneous redox state. M: marker, 1: Trebananib, 2: Leptin–Fc, 3: hGH–Fc, 4: Angiotensin–Fc, 5: Substance P–Fc, 6: Gastrin–Fc, 7: Katacalcin–Fc, S1: IgG 1 Fc region (0.19 µg), S2: IgG 1 Fc region (0.38 µg), S3: IgG 1 Fc region (0.56 µg).

Article Snippet: Genes for Trebananib fusion partner and Leptin with BamHI-EcoRI flanking sites were synthesized via codon optimization (GenScript Biotech Corp.) for E. coli expression.

Techniques: SDS Page, Mutagenesis, Marker, Purification, Produced

Journal: International Journal of Molecular Sciences

Article Title: Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli : Dissecting and Mitigating Redox Heterogeneity

doi: 10.3390/ijms232314740

Figure Lengend Snippet: Yields of mutant Fc fusion proteins (C250A, C308A) purified from 3 mL cultures (24 DWP).

Article Snippet: Genes for Trebananib fusion partner and Leptin with BamHI-EcoRI flanking sites were synthesized via codon optimization (GenScript Biotech Corp.) for E. coli expression.

Techniques: Mutagenesis, Purification