The pcDNA3.1(−)-TIM-1 and pVAX1-NS1′ were preserved in our laboratory. JEV E gene was inserted into p3×FLAG-CMV-7.1. hTIM-D114A-His, hTIM-N115A-His, and hTIM-MD were cloned into pcDNA3.1 (+). hTIM-IgVD was cloned into pCAGGS. hTIM-ECD was inserted into prokaryotic expression vectors pET-28a (+) and pGEX. Mouse anti E monoclonal antibody was kept by our lab. Mouse anti-human TIM-1 monoclonal antibody (MAB1750, R&D systems, Minneapolis, MN, USA) and goat anti-TIM-1 polyclonal antibody (AF1750, R&D systems, Minneapolis, MN, USA) were used for detecting the TIM-1 protein in Western blot and IFA. Mouse anti-His antibody (66005-1-Ig, Proteintech, Rosemont, IL, USA) was used for the enrichment of TIM-1-His protein. Mouse anti-GAPDH polyclonal antibody (sc-25778, Santa Cruz, Dallas, TX, USA) was used for Western blot. Rabbit anti-JEV E polyclonal antibody (GTX125867, GeneTex, Irvine, CA, USA), rabbit anti-JEV NS3 polyclonal antibody (GTX125868, GeneTex, Irvine, CA, USA), and rabbit anti-JEV polyclonal prM antibody (GTX131833, GeneTex, Irvine, CA, USA) were used to detected the JEV E, NS3 and prM protein, respectively, in Western blot analyses. Donkey anti-goat IgG-Alexa Fluor488 (ab150129, Abcam, Cambridge, UK), donkey anti-rabbit IgG Alexa Flour 594 (ab150076, Abcam, Cambridge, UK), and donkey anti-mouse Alexa Flour647 (ab150107, Abcam, Cambridge, UK) were used for IFA.
Gtx125868
Manufactured by GeneTex
Sourced in United States
GTX125868 is a laboratory equipment product. It serves as a core function in scientific research and analysis. The detailed description of its features and intended use is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555 goat anti rat igg, by Thermo Fisher Scientific (2 mentions)
Nb300 605, by Novus Biologicals (2 mentions)
Ab182422, by Abcam (2 mentions)
Ab133357, by Abcam (2 mentions)
Goat anti mouse igg hrp, by Thermo Fisher Scientific (2 mentions)
Mca2314ga, by Bio-Rad (2 mentions)
Nbp2 61014, by Novus Biologicals (2 mentions)
Af690, by R&D Systems (2 mentions)
Goat anti mouse igg horseradish peroxidase hrp, by Thermo Fisher Scientific (2 mentions)
Ac033, by ABclonal (2 mentions)
Ab150129, by Abcam (1 mentions)
Ab150076, by Abcam (1 mentions)
Ab150107, by Abcam (1 mentions)
Mab1750, by R&D Systems (1 mentions)
Mouse anti gapdh polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Af1750, by R&D Systems (1 mentions)
Gtx125867, by GeneTex (1 mentions)
Ripa buffer, by CoWin Biotech (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
7 protocols using gtx125868
1
Plasmid Constructs and Antibodies for JEV
2
Monoclonal Antibodies Against Japanese Encephalitis Virus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse monoclonal antibodies against E, NS1, and NS1′ of JEV were generated in our laboratory. BALB/c mice were immunized with synthetic NS1 amino acid peptide or ΔNS1′ amino acid peptide (52 amino acids generated by −1PRF). Anti-NS1 or NS1′ monoclonal antibody was screened by the hybridoma cell fusion technique and indirect enzyme-linked immunosorbent assay (ELISA). Commercial antibodies used in this study are listed as follow: JEV NS3 protein rabbit polyclonal antibody (GTX125868; GeneTex), CD11b rabbit monoclonal antibody (ab133357; Abcam), CD163 rabbit monoclonal antibody (ab182422; Abcam), MHC II (also known as SLA) mouse monoclonal antibody (MCA2314GA; Bio-Rad), iNOS rabbit polyclonal antibody (NB300-605; Novus), CD172a mouse monoclonal antibody (NBP2-61014; Novus), TNF-α goat polyclonal antibody (AF690; R&D Systems), GAPDH mouse monoclonal antibody (AC033; ABclonal), goat anti-rat IgG Alexa Fluor 555 (A-21434; Invitrogen), goat anti-rabbit IgG Alexa Fluor 488 (A-11008; Invitrogen), goat anti-mouse IgG-horseradish peroxidase (HRP) (31430; Invitrogen), and goat anti-mouse IgG-HRP (31430; Invitrogen).
3
Western Blot Analysis of NS3 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein isolation was carried out using RIPA buffer supplemented with phosphatase and protease inhibitors (CoWin Biosciences). Proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked for 1h using a solution that consisted of 5% nonfat milk diluted in Tris-buffered saline with 0.1% Tween-20 (TBST). Afterward, they were incubated overnight at 4°C with primary antibodies for NS3 (GTX125868, GeneTex) or β-actin (20536-1-AP, Proteintech). Following this, the membranes were subjected to three TBST washes before being incubated with HRP-conjugated secondary antibodies at room temperature for 1h. Lastly, protein band visualization was performed using a chemiluminescent HRP substrate (Merck Millipore, Darmstadt, Germany). Protein band imaging was conducted using the Tanon 5200 system (Tanon Science and Technology, Shanghai, China).
4
Monoclonal Antibodies Against Japanese Encephalitis Virus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse monoclonal antibodies against E, NS1, and NS1′ of JEV were generated in our laboratory. BALB/c mice were immunized with synthetic NS1 amino acid peptide or ΔNS1′ amino acid peptide (52 amino acids generated by −1PRF). Anti-NS1 or NS1′ monoclonal antibody was screened by the hybridoma cell fusion technique and indirect enzyme-linked immunosorbent assay (ELISA). Commercial antibodies used in this study are listed as follow: JEV NS3 protein rabbit polyclonal antibody (GTX125868; GeneTex), CD11b rabbit monoclonal antibody (ab133357; Abcam), CD163 rabbit monoclonal antibody (ab182422; Abcam), MHC II (also known as SLA) mouse monoclonal antibody (MCA2314GA; Bio-Rad), iNOS rabbit polyclonal antibody (NB300-605; Novus), CD172a mouse monoclonal antibody (NBP2-61014; Novus), TNF-α goat polyclonal antibody (AF690; R&D Systems), GAPDH mouse monoclonal antibody (AC033; ABclonal), goat anti-rat IgG Alexa Fluor 555 (A-21434; Invitrogen), goat anti-rabbit IgG Alexa Fluor 488 (A-11008; Invitrogen), goat anti-mouse IgG-horseradish peroxidase (HRP) (31430; Invitrogen), and goat anti-mouse IgG-HRP (31430; Invitrogen).
5
JEV Infection Molecular Mechanisms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Various commercially available antibodies were used in this study. Antibodies against LC3B (#3868), Beclin1 (#3495) and GAPDH (#2118) were obtained from Cell Signaling Technologies (Danvers, MA). The antibody against JEV NS3 (GTX125868) was from GeneTex (Irvine, CA). The antibody against Nedd4 (ab14592) was purchased from Abcam (Cambridge, MA). HRP-conjugated secondary antibodies against mouse (Cat #: 31430) or rabbit (Cat #: 31460) IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG (Cat #: A32723) and Alexa Fluor 555-conjugated goat anti-rabbit IgG (Cat #: A32732) were purchased from Life Technologies (InvitrogenTM, Carlsbad, CA). Cyclosporin A (#S2286) and rapamycin (#S1039) were purchased from Selleck Chemicals (Houston, TX, USA). All of the plasmids used for JEV pseudotyped virus (JEVpv) generation were kindly provided by Yoshiharu Matsuura (Osaka University, Japan). A cDNA encoding the full-length human Nedd4 was cloned into the pcDNA3.1 vector. All constructs were confirmed by sequencing.
6
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in lysis buffer containing radioimmunoprecipitation assay (RIPA) buffer, phenylmethanesulfonyl fluoride (PMSF), sodium orthovanadate (Na3VO4), and a protease inhibitor mixture. Equal quantities of proteins were electrophoretically separated on 10% sodium dodecyl sulfate‒polyacrylamide gels (SDS‒PAGE) and transferred to nitrocellulose membranes (Pall Corp., USA). The membranes were incubated with 5% skim milk in Tris-buffered saline (TBS) buffer with 0.1% Tween-20 for 1 h at room temperature (RT). Subsequently, the membranes were incubated with the indicated primary antibodies for 2 h and with secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h. Immunoreactive bands were developed using enhanced chemiluminescence reagents (LF-QC0103, AbFrontier Inc.). We used primary antibodies against NS3 (GTX125868, GeneTex), phospho-Bcl10 (sc-81,484, Santa Cruz Biotechnology), phospho-NFκB, LC3B, P62 (#3033, #4108, and #5114, Cell Signaling Technology), and β-actin (A5441, Sigma–Aldrich).
7
Western Blot Analysis of Viral Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA lysis buffer (P0013B, Beyotime, China) and 1 mM PMSF (ST506, Beyotime, China). Protein concentration of cell lysate was determined by the BCA method (Pierce, Rockford, USA). Ten micrograms of total protein per sample was loaded onto sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 3–4 h and transferred to PVDF membrane at 350 mA for 90 min (Version8, Roche, USA) using an electro-blotting method. After incubating in blocking buffer (PBST with 1% (w/v) BSA (A7030, Sigma)) for 1 h, membranes were incubated with rabbit polyclonal antibody for NS3 (GTX125868, Genetex, USA), rabbit polyclonal antibody for SP1 (ab13370, Abcam, USA) at 4 °C for 12 h. After primary antibodies were used, the membranes were washed before Horseradish Peroxidase (HRP)-conjugated Goat anti-rabbit IgG second-antibody (sc-2030, Santa Cruz, USA) was added for 1 h at room temperature and washed again. The membranes were visualized with an ECL Western blot detection kit (NC15080, Thermo). The β-actin (#4970, Cell Signalling Technology, USA) protein level was also examined as an internal control. The chemiluminescence intensity of each protein band was quantified using the Image J software, and then protein levels were normalized by the amount of β-actin protein.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!