rabbit polyclonal antibodies against flag tag  (Proteintech)

Journal: Journal of Virology

Article Title: The “LLQY” Motif on SARS-CoV-2 Spike Protein Affects S Incorporation into Virus Particles

doi: 10.1128/jvi.01897-21

Figure Lengend Snippet: Tyrosine residue 756 is critical for the cleavage of the prefusion S protein. (A) Schematic of the domain structure of SARS-CoV-2 spike proteins and amino acid sequence alignment of the S1/S2 (R685) and S2′ (R815) cleavage site and the region between them in S2 subunit with the corresponding fragments of SARS-CoV and MERS-CoV. (B to D) The hydrogen bonds formed by Y756 and the leucine at position 753 (L753), the phenylalanine at position 759 (F759), or the threonine residue at position 998 (T998) in the folded S trimer can be observed in the cryo-EM structure of the S ectodomain trimer (PDB code 6VSB ). (E) Western blot analysis of the effect of Y756, R685, or R815 mutation on the expression and processing of SARS-CoV-2 spike proteins using anti-RBD (S1) and Flag (C-terminal of S2). GAPDH was used as a loading control. (F) Western blot analysis of the effect of Y756 mutation to different amino acids on the synthesis and processing of S protein using anti-RBD and anti-Flag antibodies. GAPDH was used as a loading control. (G) A summary diagram of the regulation of S protein cleavage when Y756 is mutated to phenylalanine (F), tryptophan (W), histidine (H), cysteine (C), alanine (A), glycine (G), or glutamate (E). (H) Western blot analysis of the effect of L753, F759, or T998 mutation on the synthesis and processing of S protein using anti-RBD and anti-Flag antibodies. GAPDH was used as a loading control.

Article Snippet: Rabbit polyclonal antibodies against Flag ( DYKDDDDK ) tag (no. 20543-1-AP), ACE2 (no. 21115-1-AP), GAPDH (no. 10494-1-AP), and β-actin (no. 20536-1-AP), and CoraLite 488-conjugated ACE2 monoclonal antibody (no. CL488-66699) were purchased from Proteintech (China).

Techniques: Residue, Sequencing, Cryo-EM Sample Prep, Western Blot, Mutagenesis, Expressing, Control

Journal: Journal of Molecular Cell Biology

Article Title: Host HDAC4 regulates the antiviral response by inhibiting the phosphorylation of IRF3

doi: 10.1093/jmcb/mjy035

Figure Lengend Snippet: HDAC4 interacts with TBK1, IKKε, and IRF3. (A) IP and IB analyses of lysates of HEK293T cells overexpressing vector (VC) or plasmids encoding Flag-tagged RIG-I, MAVS, TBK1, IKKε, and IRF3, plus HA-tagged HDAC4, probed with anti-HA or anti-Flag. Bottom, IB analysis of lysates without IP (10% of input). (B and C) IP and IB analyses of lysates of HEK293T cells overexpressing plasmids encoding HA-tagged IKKε or HA-tagged TBK1, HA-tagged HDAC4, and Flag-tagged IRF3, probed with various combinations of anti-HA or anti-Flag. (D and E) Immunoassay of lysates of HEK293T cells (5 × 106) left uninfected or infected for 8 h with SeV, followed by IP analysis with immunoglobulin G (IgG), as a control (first lane), or with antibody to HDAC4 or TBK1 and IB analysis with antibodies to IKKε and IRF3 (D) or HDAC4 and IRF3 (E). Bottom, IB analysis of the samples above (input) without IP. (F) The endogenous association of TBK1, IKKε, and IRF3 in the presence or absence of HDAC4. IP analysis with anti-IRF3 and IB analysis with anti-IRF3, anti-TBK1, anti-IKKε, anti-HDAC4, or anti-β-actin of HEK293T cells infected with SeV for 6 h. Data are representative of three independent experiments.

Article Snippet: Rabbit polyclonal antibody against Flag (#2368S) and rabbit monoclonal antibodies against HA (#3724), HDAC4 (#7628), HDAC4 phospho-S246 (#3443), Lamin B1 (#13435), β-Tubulin (#2128), TBK1 (#3013S), IKKε (#2905), and IRF3 phospho-S396 (#29047) were all from Cell Signaling Technology.

Techniques: Plasmid Preparation, Infection

Journal: Journal of Molecular Cell Biology

Article Title: Host HDAC4 regulates the antiviral response by inhibiting the phosphorylation of IRF3

doi: 10.1093/jmcb/mjy035

Figure Lengend Snippet: HDAC4 interacts with TBK1/IKKε via the TBK1/IKKε kinase domain and HDAC4 impairs the phosphorylation of IRF3 by TBK1/IKKε. (A and B) IB analysis of HEK293T cells transiently cotransfected for 48 h with Flag-tagged HDAC4 and EGFP-tagged wild-type TBK1, HA-tagged wild-type IKKε, or their mutants, assessed with whole-cell lysates (10% input) or IP with anti-Flag antibody. (C and D) In vitro kinase assay of IRF3 phosphorylated at Ser386 or Ser396 and HDAC4 phosphorylated at Ser246 or other serine or threonine sites (pSer/Thr). The peptide of IRF3 full-length protein at its carboxyl terminus to GST and recombinant protein IKKε or TBK1 with kinase activity were introduced into a mixture containing overexpressed and immunoprecipitated Flag-tagged HDAC4.

Article Snippet: Rabbit polyclonal antibody against Flag (#2368S) and rabbit monoclonal antibodies against HA (#3724), HDAC4 (#7628), HDAC4 phospho-S246 (#3443), Lamin B1 (#13435), β-Tubulin (#2128), TBK1 (#3013S), IKKε (#2905), and IRF3 phospho-S396 (#29047) were all from Cell Signaling Technology.

Techniques: In Vitro, Kinase Assay, Recombinant, Activity Assay, Immunoprecipitation

Journal: Biological Procedures Online

Article Title: Fluorescence ImmunoPrecipitation (FLIP): a Novel Assay for High-Throughput IP

doi: 10.1186/s12575-016-0046-x

Figure Lengend Snippet: Evaluation of the amount of cells necessary for reliable FLIP. HeLa cells were plated, transfected and lysed in wells of different sizes. Lysates from each well were used to perform FLIP and IP/IB. a FLIP signal (total mean fluorescence measured by ImageJ) using a FLAG Ab subtracted from the FLIP signal using control IgG antibodies (background) is reported. Two FLIP measurements from 2 different aliquots of beads were used to calculate the range of variability of the measurement (var.). b The immunoprecipitated beads not used for FLIP were analyzed by immunoblotting. LY = input lysate; IgG = control IP using IgG antibodies; FLAG = IP of the tagged protein using anti FLAG antibodies. Tubulin was used as loading control

Article Snippet: After SDS-PAGE and transfer of the proteins onto a PVDF-F membrane, proteins were probed with a rabbit polyclonal antibody against FLAG tag (Cell Signaling #2368) mixed with the test mouse antibody.

Techniques: Transfection, Fluorescence, Control, Immunoprecipitation, Western Blot