immunoprecipitation with myc  (MedChemExpress)

Journal: mBio

Article Title: Quantitative proteomic analysis identifies the unfolded protein response as a host pathway co-opted by ASFV to promote replication

doi: 10.1128/mbio.03242-25

Figure Lengend Snippet: D117L activates three UPR pathways through targeting key UPR components. ( A ) Heatmap of ASFV proteins identified by proteomics analysis across infection. ( B ) PPI network analysis of the virus-host interaction. ( C ) HEK293T cells were transfected with vector or Flag-D117L for 24 h, and mRNA expression of Ddit3, Dnajc3, and Dnajb9 was detected by RT-qPCR. ( D ) HEK293T cells were transfected with vector, Flag-D117L, Flag-D117LΔ38-60, Flag-D117LΔ67-76, or Flag-D117LΔ96-117; each group was also co-transfected with ATF6-LUC and Renilla-TK. After 24 h post-transfection, luciferase activities were examined. ( E ) Mass spectrometry was performed to identify the interacting proteins of D117L and D117LΔ38-60, and the top 10 proteins were listed. ( F ) Kyoto Encyclopedia of Gene and Genomes pathway enrichment analysis of D117L interacting proteins. ( G and H ) Immunofluorescence analysis of the colocalization of Flag-tagged D117L (green) and Myc-tagged or endogenous HSPA5 (red) in iPAM cells. ( I ) Immunoprecipitation (IP) assay examining the interaction between HSPA5 and D117L. ( J and K ) IP assays assessing the interaction of D117L (WT) or D117LΔ38-60, D117LΔ67-76, and D117LΔ96-117 mutants with CANX or HSPA5 in HEK293T cells.Data are presented as mean + SD. PP values were calculated by a two-tailed unpaired t-test. ** P < 0.01, *** P < 0.001.

Article Snippet: For immunoprecipitation analysis, the HEK293T cells were homogenized with IP lysis buffer (containing 50 mM Tris-Cl [pH = 7.4], 150 mM NaCl, 1%Triton-100, 0.1% SDS, 1% sodium deoxycholate, and protease inhibitor cocktail), centrifuged to remove cell debris, and then the supernatant was collected for immunoprecipitation with Myc (HY-K0206, MCE) and DYKDDDDK magnetic beads (b26103; Selleck Chemicals, USA) at 4°C for 2 h. After washing five times with IP lysis buffer, the protein-bound beads were finally resuspended in SDS-PAGE loading buffer.

Techniques: Infection, Virus, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Luciferase, Mass Spectrometry, Immunofluorescence, Immunoprecipitation, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Evolutionary-Distinct Viral Proteins Subvert Rice Broad-Spectrum Antiviral Immunity Mediated by the RAV15-MYC2 Module.

doi: 10.1002/advs.202412835

Figure Lengend Snippet: Figure 1. Distinct viral proteins interacted with OsRAV15. A) Y2H assays showing the interaction between OsRAV15 and different viral proteins (RSV P2, SRBSDV SP8, and RSMV M) in yeast cells. Viral proteins were cloned into pGBKT7 (BD), while OsRAV15 was cloned into the pGADT7 (AD) yeast vector. The different combinations were transformed into yeast cells and grown on SD-L-T plates at 30 °C for 3 days. Colony growth was scanned after 3 days of incubation in SD-L-T-H-Ade medium. B–D) Co-immunoprecipitation (Co-IP) assays showing that OsRAV15 interacted with viral proteins P2 (B), SP8 (C), and M (D) in vivo. OsRAV15-MYC and P2-FLAG, SP8-GFP, M-FLAG, or GFP-FLAG (negative control) were transiently co-expressed in N. benthamiana leaves. Total proteins were extracted, and the supernatants were precipitated with FLAG or GFP beads, followed by Co-IP. The immunoprecipitated (IP) and input proteins were then analyzed using anti-MYC and anti-GFP or anti-FLAG antibodies. The red asterisks represent the specific band. E) BiFC assays confirming the interactions of OsRAV15 with viral proteins P2, SP8, and M. cYFP-OsRAV15 was co-expressed with P2-nYFP, SP8-nYFP, M-nYFP or the negative control Gus-nYFP into N. benthamiana leaves. The white dotted box is an enlarged area. The images were captured by confocal microscopy at 48 hpi. Scale bar = 50 μm. F) Co-IP assays of proteins isolated from healthy OsRAV15 and RSV-infected OsRAV15 plants by RSV P2 and MYC antibody. The red asterisks represent the specific band. G) Co-IP assays of proteins isolated from healthy OsRAV15 and RSMV-infected OsRAV15 plants by RSMV M and MYC antibody. The red asterisks represent the specific band. The precipitated proteins were analyzed by western blots with the indicated antibodies. Each experiment was repeated three times with similar results.

Article Snippet: The protein was extracted using extraction buffer (Thermo Scientific, Cat. no. 87788) containing 10 mm DTT, 1 × EDTAfree protease inhibitor cocktail (Roche, Basel, Switzerland) for 30 min at 4° C. Immunoprecipitation assays were performed using Pierce anti-c-Myc magnetic beads (Thermo Scientific, USA), anti-FLAG M2 beads (Sigma– Aldrich, USA), anti-GFP-trap beads (Chromotek, Germany) and Protein A/G OsMYC2 antibody beads, respectively for ≈2 h at 4° C (with gentle shaking).

Techniques: Clone Assay, Plasmid Preparation, Transformation Assay, Incubation, Immunoprecipitation, Co-Immunoprecipitation Assay, In Vivo, Negative Control, Confocal Microscopy, Isolation, Infection, Western Blot

Journal: iScience

Article Title: Revisiting phosphoregulation of Cdc25C during M-phase induction

doi: 10.1016/j.isci.2024.111603

Figure Lengend Snippet:

Article Snippet: anti-myc for immunoprecipitation (IP) , Cell Signaling Technology , Cat# 2276; RRID: AB_331783.

Techniques: Western Blot, Immunoprecipitation, Sequencing, Modification, Mass Spectrometry, Recombinant, Protease Inhibitor, Mutagenesis, Purification, Expressing