co immunoprecipitation hek293t  (ATCC)

Journal: Nature biotechnology

Article Title: A comprehensive SARS-CoV-2–human protein–protein interactome reveals COVID-19 pathobiology and potential host therapeutic targets

doi: 10.1038/s41587-022-01474-0

Figure Lengend Snippet: a, UpSet plot showing the overlap of SARS-CoV-2–human protein–protein interactions from four studies (Supplementary Table 3). Each bar shows the interactions shared by only the marked studies at the bottom. Composition of each bar in terms of the source of the interactions are indicated by different colors. b, Co-IP confirming ORF3a–ZNF579 interaction in HEK293T cells following transfection with ORF3a–FLAG or empty vector. The experiment was repeated independently three times with similar results. c, Western blot showing levels of ZNF579 along with GAPDH as a loading control in HEK293T cells following transfection with ORF3a–FLAG or empty vector. Experiment was repeated independently three times with similar results. d, ChIP–seq for ZNF579 in MCF7 cells from the ENCODE consortium at the HSPA6 locus. Signal is log2FC over input. e, Expression of HSPA6 after transfection with ORF3a–FLAG or empty vector (E.V.). Two transfection replicates were probed with two primer pairs to HSPA6 at three different template dilutions in technical triplicate (18 total reactions for each condition). Expression is normalized to GAPDH and then to the empty vector average using the ΔΔCt method. Box plots display the median as the center line, the 1st and 3rd quartiles as hinges and 1.5× interquartile range as whiskers. Significance was assessed using the two-tailed Mann Whitney U test. f,g Co-IP confirming ORF7b–STT3A and ORF7b–Sec61 interactions in HEK293T cells following transfection with ORF7b–FLAG or empty vector and STT3A–MYC or Sec61–V5, respectively. Each experiment was repeated independently three times with similar results. h, Co-IP confirming N-histone H1.4 interaction in HEK293T cells following transfection with N or empty vector and histone H1.4. Experiment was repeated independently three times with similar results.

Article Snippet: Co-immunoprecipitation HEK293T (CRL-3216; ATCC) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (catalog no. 30-2002; ATCC) supplemented with 10% FBS (catalog no. 30-2020; ATCC) and incubated at 37 °C with 5% CO 2 .

Techniques: Protein-Protein interactions, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Western Blot, Control, ChIP-sequencing, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Biochemical and biophysical research communications

Article Title: CaMKIIα Phosphorylation of Shank3 Modulates ABI1-Shank3 Interaction

doi: 10.1016/j.bbrc.2020.01.089

Figure Lengend Snippet: A, B. Lysates of HEK293T cells expressing HA-ABI1 (A) or HA-CaV1.3-CTD (B) and either empty vector, GFP-Shank3-WT, GFP-Shank3-S685A, or GFP-Shank3-S685D were immunoprecipitated (IP) using a GFP antibody. Input samples and IP complexes were resolved by SDS-PAGE and immunoblotted for GFP-Shank3 and HA-ABI1 (A) or HA-CaV1.3-CTD (B). HA/GFP signals for each IP lane were calculated and normalized to GFP-Shank3-WT. Both GFP-Shank3–685A and GFP-Shank3–685D significantly reduce HA-ABI1 co-immunoprecipitation (GFP-Shank3-S685A: 37±11% reduced compared to WT, ** p < 0.01, GFP-Shank3-S685D: 32±8% reduced compared to WT, *** p < 0.001, one sample Student’s t-test with equal variance compared to theoretical mean of 100). Mutation of S685 had no effect on HA-CaV1.3-CTD co-immunoprecipitation (one sample Student’s t-test with equal variance compared to theoretical mean of 100). The immunoblots shown are representative of 4–5 biological replicates that were quantified. Error bars, mean ± SEM.

Article Snippet: Cell Culture and Co-Immunoprecipitation HEK293T cells (ATCC catalog CRL-3216) were cultured and maintained in DMEM containing 10% FBS and 1% penicillin/streptomycin at 37°C with 5% CO 2 .

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, SDS Page, Mutagenesis, Western Blot

Journal: Biochemical and biophysical research communications

Article Title: CaMKIIα Phosphorylation of Shank3 Modulates ABI1-Shank3 Interaction

doi: 10.1016/j.bbrc.2020.01.089

Figure Lengend Snippet: Left, GST, GST-Shank3 WT, or GST-Shank3-S685A were pre-phosphorylated with CaMKIIα and incubated with HEK293T cell lysate expressing HA-ABI1. Control protein samples were pre-incubated in the absence of CaMKIIα. Isolated GST complexes were then analyzed by immunoblotting as indicated (see Methods). Immunoblots are representative of 5 independent experiments. Right, HA/GST signals from each pulldown lane were normalized to GST-Shank3-WT/no CaMKII condition. Incubation with CaMKIIα significantly increases HA-ABI1 binding to GST-Shank3-WT (148±14% increased relative to no CaMKII condition, * p < 0.05, one sample Student’s t-test with equal variance compared to theoretical mean of 100), which is not observed with GST-Shank3-S685A (unpaired Student’s t-test with equal variance). Error bars, mean ± SEM.

Article Snippet: Cell Culture and Co-Immunoprecipitation HEK293T cells (ATCC catalog CRL-3216) were cultured and maintained in DMEM containing 10% FBS and 1% penicillin/streptomycin at 37°C with 5% CO 2 .

Techniques: Incubation, Expressing, Control, Isolation, Western Blot, Binding Assay

Journal: Cancer research

Article Title: Asporin Restricts Mesenchymal Stromal Cell Differentiation, Alters the Tumor Microenvironment, and Drives Metastatic Progression

doi: 10.1158/0008-5472.CAN-18-2931

Figure Lengend Snippet: ASPN binds to BMP-4 and restricts BMP-4-induced signaling. A, GSEA of TGF-β family pathway genes in Aspn+/+ and Aspn−/− fetal MSCs as determined from microarray (n=3). B, C, BMP-4-induced signaling in Aspn+/+ and Aspn−/− fetal MSCs (A and B represent independently derived MSCs) as measured by immunoblotting and quantified using ImageJ. Aspn+/+ and Aspn−/− fetal MSCs were serum-depleted and then incubated with recombinant mouse BMP-4 (5ng/mL) for 30 minutes. Statistical analyses performed using Student’s t-test (n≥3). D, E, Recombinant mouse ASPN restricts BMP-4-induced signaling in Aspn−/− fetal MSCs as measured by immunoblotting and quantified using ImageJ. Aspn+/+ and Aspn−/− fetal MSCs were serum-depleted and then incubated with recombinant mouse BMP-4 (5ng/mL) with vehicle or recombinant mouse ASPN (100ng/mL) for 30 minutes. Statistical analyses performed using Student’s t-test (n≥2). F, G, BMP-4-induced signaling in WPMY-1-Neo, WPMY-1-ASPN D13, and WPMY-1ASPN D14 (A and B represent independently derived clones) as measured by immunoblotting and quantified using ImageJ. WPMY-1-Neo, WPMY-1-ASPN D13, and WPMY-1ASPN D14 were serum-depleted and then incubated with recombinant human BMP-4 (5ng/mL) for 30 minutes. Statistical analyses performed using one-way ANOVA with Tukey multiple comparison (n≥3). H, Co-immunoprecipitation of ASPN and BMP-4. FLAG tagged mouse ASPN was transfected in HEK293T cells and then incubated with recombinant mouse BMP-4. ASPN was immunoprecipitated with anti-FLAG beads and then examined by immunoblotting for ASPN and BMP-4 (n=2). I, Aspn+/+ and Aspn−/− fetal MSCs were cultured in osteogenic-inducing media with either vehicle, 250nM LDN-193189, or recombinant mouse BMP4 (5ng/mL) and then stained for Alizarin Red. J, Aspn+/+ and Aspn−/− fetal MSCs were cultured in osteogenic-inducing media with either vehicle, 250nM LDN-193189, or recombinant mouse BMP4 (5ng/mL) and then examined for osteogenic-induced genes by qRT-PCR. Statistical analyses performed using one-way ANOVA with Tukey multiple comparison (n≥3). Graphs shown as mean±SEM, *P≤0.05, **P≤0.01, ***P≤0.001, and black bars=100μM.

Article Snippet: Co-immunoprecipitation assays HEK293T cells were transfected with a murine ASPN-Myc-FLAG tagged expression vector (Origene) or vehicle control using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Microarray, Derivative Assay, Western Blot, Incubation, Recombinant, Clone Assay, Immunoprecipitation, Transfection, Cell Culture, Staining, Quantitative RT-PCR