recombinant human grp94 protein  (RayBiotech inc)

Journal: Cell Reports

Article Title: Fibroblast-Derived STC-1 Modulates Tumor-Associated Macrophages and Lung Adenocarcinoma Development

doi: 10.1016/j.celrep.2020.107802

Figure Lengend Snippet: Extracellular STC1 Inhibits IMC Differentiation (A) Confocal microscopy (Mac2-IF) and differential interference contrast (DIC) imaging of STC1 +/+ IMCs treated with 50% mock- or STC1-CM for 5 days in the presence of 5% fetal bovine serum (FBS). DMEM culture served as a negative control. Scale bars, 20 μm. (B) STC1 +/+ IMCs treated as in (A) were analyzed for F4/80 and CD11b expression by flow cytometry. The left bar graph shows F4/80 + and CD11b + percentages (n = 3). Representative flow cytometry plots are shown on the right. (C) Identification of STC1-binding secretory proteins. STC1-GFP protein secreted by the transfected HEK293 T cells was immunoprecipitated using GFP-trap (left, “bound” fraction) and resolved on SDS-PAGE to visualize STC1-associated proteins by CBB staining (middle). Five candidate proteins with signal peptides were identified by mass spectrometry analysis of the excised protein bands (right). (D) Co-immunoprecipitation of secreted endogenous STC2 or GRP94 with His-tagged STC1. CM of HEK293 T cells transfected with His-tagged STC1 (STC1-CM) were immunoprecipitated for the His-tag (His-tag-IP), followed by STC1, STC2 or GRP94 immunoblotting.

Article Snippet: Recombinant human GRP94 protein , RayBiotech , Cat# 228-21002-2.

Techniques: Confocal Microscopy, Imaging, Negative Control, Expressing, Flow Cytometry, Binding Assay, Transfection, Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Western Blot

Journal: Cell Reports

Article Title: Fibroblast-Derived STC-1 Modulates Tumor-Associated Macrophages and Lung Adenocarcinoma Development

doi: 10.1016/j.celrep.2020.107802

Figure Lengend Snippet: Extracellular STC1 Interferes with GRP94 (A) Intracellular GRP94 protein expression in primary cultures of fractionated IMCs, AT2, and fibroblasts (Fib) populations from Stc1-WT/KO BVE mice, as determined by immunoblotting. ERK2 blots served as loading controls. (B) Immunoblot detection of GRP94 secreted into CM during 3 days of culture of IMC and non-IMC populations from Stc1-WT/KO BVE mice. Total protein (CBB) staining served as the loading control. (C) Confocal imaging of Stc1-WT/KO IMCs for GRP94/STC1 immunofluorescence. STC1-transfected HEK293 T cells treated with BD GolgiStop served as positive controls for STC1 staining. Scale bars, 10 μm. (D) Stc1 +/+ IMCs cultured for 72 h in IMC-CM depleted for GRP94 were evaluated by flow cytometry for F4/80 induction. The efficacy of immunodepletion was confirmed by GRP94 immunoblotting on the left. Representative F4/80 plots are indicated in the middle along with %F4/80 + and F4/80 MFI. The bar graph in the right shows %F4/80 reduction by GRP94 depletion (n = 3). (E) Scavenger receptor expression in IMCs. Confocal imaging of Stc1-WT/KO IMCs stained for SR-A1 and Mac2 (left). Maximum intensity Z projection images are indicated. Scale bars, 20 μm. SR-F1 immunoblotting of Stc1-WT/Het/KO IMC lysates (right). ERK2 blots served as loading controls. (F) Fucoidan-induced IMC maturation in the presence of STC1. %F4/80 + cells (left) and %CD11b high F4/80 high cells (right) were measured after 48 h of incubation ± fucoidan in 50% STC1-CM. (G) ELISA assay quantitation of recombinant GRP94 binding to immobilized SR-A1 in the presence of 100 ng/mL recombinant STC1 (n = 3).

Article Snippet: Recombinant human GRP94 protein , RayBiotech , Cat# 228-21002-2.

Techniques: Expressing, Western Blot, Staining, Imaging, Immunofluorescence, Transfection, Cell Culture, Flow Cytometry, Incubation, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Recombinant, Binding Assay

Journal: Cell Reports

Article Title: Fibroblast-Derived STC-1 Modulates Tumor-Associated Macrophages and Lung Adenocarcinoma Development

doi: 10.1016/j.celrep.2020.107802

Figure Lengend Snippet:

Article Snippet: Recombinant human GRP94 protein , RayBiotech , Cat# 228-21002-2.

Techniques: Plasmid Preparation, Recombinant, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Software, Imaging, Mass Spectrometry