Cells were lysed using 4 × SDS loading buffer and denatured at 95 °C for 10 min. Protein samples were resolved by SDS-PAGE, transferred to PVDF membranes (GE Healthcare), and processed for western blotting. Western blot detection of MYH9 was performed using a rabbit anti-MYH9 antibody (1:1,000, A0173, Abclonal), with a goat anti-rabbit IgG-HRP antibody (1:3,000, B2615, Santa Cruz Biotechnology) as the secondary antibody. Other antibodies used in the study included: mouse anti-Flag M2 (1:2,000, F1804, Sigma), rabbit anti-Lamin B1 (D4Q4Z) mAb (1:3,000, 12,586, Cell Signaling Technology), mouse anti-GAPDH (1:3,000, AC002, Abclonal), rabbit anti-Influenza A virus NP antibody (1:2,000, GTX125989, GeneTex), rabbit anti-Influenza A virus M1 antibody (1:2,000, GTX125928, GeneTex), rabbit anti-Influenza A virus PA antibody (1:2,000, GTX125932, GeneTex), rabbit anti-Influenza A virus PB1 antibody (1:2,000, GTX125923, GeneTex), rabbit anti-Influenza A virus PB2 antibody (1:2,000, GTX125926, GeneTex), goat anti-rabbit IgG-HRP (1:5,000, B2615, Santa Cruz Biotechnology), and goat anti-mouse IgG-HRP (1:5,000, 31,430, Invitrogen) antibodies. GAPDH was used as a loading control. Protein bands were visualized by either fluorescence or using a chemiluminescent ECL substrate (GE Healthcare).
Gtx125923
Manufactured by GeneTex
Sourced in United States
GTX125923 is a laboratory equipment product from GeneTex. It is designed for general research applications.
Automatically generated - may contain errors
Lab products found in correlation
Gtx125989, by GeneTex (7 mentions)
Gtx125926, by GeneTex (5 mentions)
Irdye 680 goat anti rat, by LI COR (4 mentions)
Gtx125928, by GeneTex (3 mentions)
Gtx125932, by GeneTex (3 mentions)
Mca77g, by Bio-Rad (3 mentions)
Donkey anti rabbit irdye 800, by LI COR (3 mentions)
Anti tubulin, by Bio-Rad (2 mentions)
Gtx118991, by GeneTex (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Goat anti mouse igg hrp, by Thermo Fisher Scientific (1 mentions)
A0173, by ABclonal (1 mentions)
B2615, by Santa Cruz Biotechnology (1 mentions)
Ecl chemiluminescent substrate, by GE Healthcare (1 mentions)
F1804, by Merck Group (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Ac002, by ABclonal (1 mentions)
Ab92824, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
10 protocols using gtx125923
1
Western Blot Analysis of Influenza Proteins
2
IAV Protein Detection and Subcellular Fractionation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IAV proteins were detected using polyclonal anti-IAV PB1 protein antibody (GTX125923, GeneTex) and anti-IAV NP protein antibody (GTX125989, GeneTex), diluted 1:2000 in phosphate buffered saline (PBS)/5% BSA (RPI). To confirm fractionation of the A549 cells, Mitotracker (AB92824, Abcam), γ-tubulin (MCA77G, Bio-rad) and Histone H3 (AB1791, Abcam) antibodies were used, diluted 1:1000–5000 in PBS/5% BSA (RPI). Secondary antibodies IRDye 800 goat anti-rabbit (926-32211, LI-COR), and IRDye 680 goat anti-rat (926-68076, LI-COR) diluted 1:10,000 in PBS/5% BSA were used. Western blots were developed using the Odyssey CLx imaging system (LI-COR).
3
Detecting Influenza A Virus Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IAV proteins were detected using rabbit polyclonal antibodies anti-PB1 (GTX125923, GeneTex), and anti-NP (GTX125989, GeneTex) diluted 1:2000 in blocking buffer (PBS, 5 per cent bovine serum albumin Research Products International (RPI), 0.1 per cent Tween 20 (RPI)). Cellular proteins were detected using the rat monoclonal antibody anti-tubulin (MCA77G, Bio-Rad) diluted 1:3000 in blocking buffer. Secondary antibodies IRDye 800 donkey anti-rabbit (926-32213, LI-COR) and IRDye 680 goat anti-rat (926-68076, LI-COR) were used to detect Western signals with a LI-COR Odyssey scanner.
4
Detecting IAV Proteins by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IAV proteins were detected using rabbit polyclonal antibodies anti-PB1 (GTX125923, GeneTex), and anti-NP (GTX125989, GeneTex) diluted 1:2000 in blocking buffer (PBS, 5% bovine serum albumin (RPI), 0.1% Tween-20 (RPI)). Cellular proteins were detected using the rat monoclonal antibody anti-tubulin (MCA77G, Bio-Rad) diluted 1:3000 in blocking buffer. Secondary antibodies IRDye 800 donkey anti-rabbit (926-32213, LI-COR) and IRDye 680 goat anti-rat (926-68076, LI-COR) were used to detect Western signals with a LI-COR Odyssey scanner.
5
Influenza Viral Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293-T cells were transfected with pCAGGs plasmids encoding for PA, PB2, PB1 (WT or mutant), 24 h p.t. the cells were lysed using ice-cold radio immunoprecipitation assay buffer (RIPA; 150 mM NaCl, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 25 mM TRIS (pH 7.5), 1% (v/v) Triton X-100, protease inhibitor cocktail). Clarified cell lysates were adjusted to equal amounts using Pierce™ BCA assay (Thermo Fisher), mixed with Laemmli buffer (4X), incubated at 95 °C for 5 min, and subjected to SDS-PAGE and western blot. Proteins were detected using the primary antibodies rabbit anti-PB1 (GTX125923, Genetex; 1:2000 in blocking buffer), rabbit anti-PA (GTX125932, Genetex; 1:1000 in blocking buffer), and mouse anti-Tubulin (clone DM1A, Sigma-Aldrich; 1:1000 in blocking buffer) and the secondary antibody anti-Rabbit IgG-800CW (LI-COR; 1:10,000 in blocking buffer), and anti-Mouse IgG-680RD (LI-COR; 1:10,000 in blocking buffer). Uncropped blots are provided in a source data file.
6
Antibody-based Immunodetection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used in the study included the following: rabbit anti-TRA2A (GTX87998, GeneTex, USA); mouse anti-Flag (F1804, Sigma-Aldrich, Saint Louis, MO, USA); mouse anti–HA-tag (M180-3, MBL, Japan); mouse anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CB100127, California Bioscience, Coachella, CA, USA); rabbit anti-IAV PA, PB1, PB2, NP, NS1, M1, NEP, M2, HA (GTX118991, GTX125923, GTX125926, GTX125989, GTX125990, GTX125928, GTX125953, GTX125951, and GTX127357, GeneTex, USA); mouse anti-NP (produced in our laboratory); control rabbit IgG polyclonal (AC005, ABclonal Biotechnology, Cambridge, MA, USA); horseradish peroxidase–conjugated anti-mouse and anti-rabbit (BF03001 and BF03008, Beijing Biodragon Inmmunotechnologies, China); Alexa Fluor 594-conjugated goat anti-rabbit (GR200G-43C, Sungene Biotech); fluorescein isothiocyanate (FITC)–goat anti-mouse (GM200G-02C, Sungene Biotech); and FITC-goat anti-rabbit (GR200G-02C, Sungene Biotech). 4′,6′-diamidino-2-phenylindole (1:1000) (no. C1002) was purchased from the Beyotime, China.
7
Influenza Polymerase Subunit Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A549 cells were seeded on glass coverslips and transfected with pCAGGs plasmids expressing either WT or mutants of PB2, PB1 (in combination with nHA-PA), and PA (in combination with nHA-PB1)74 (link) using XtremeGeneTM (Roche) 24 h p.t., cells were fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 3% BSA in PBS (blocking buffer). For immunostaining coverslips were incubated with the primary antibodies rabbit anti-PB2 (GTX125926; Genetex; 1:3000 in blocking buffer), rabbit anti-PB1 (GTX125923, Genetex; 1:3000 in blocking buffer), rabbit anti-PA (GTX125932, Genetex; 1:3000 in blocking buffer), rat anti-HA-Tag (clone 3F10, Roche; 1:500 in blocking buffer), overnight at 4 °C and with secondary antibodies anti-rabbit Alexa Fluor 488 (Invitrogen; 1:2000 in blocking buffer) or anti-rabbit Alexa Fluor 568 (Invitrogen; 1:2000 in blocking buffer) and anti-rat Alexa Fluor 488 (Invitrogen; 1:2000 in blocking buffer) for 1 h at room temperature. Cell nuclei were stained with DAPI (Thermo Fisher Scientific) for 20 min at room temperature. Coverslips were mounted using Mounting Medium S3023 (Dako Omnis) and examined using an LSM-800 Airyscan confocal microscope (Carl Zeiss) and ZEN software (v2.6).
8
Visualizing Influenza PB1 Protein Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The localizations of wild-type PB1 and mutant PB1 proteins were determined by Immunofluorescence analysis (IFA). Infected A549 cells were fixed in 2% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then blocked with 3% bovine serum albumin and 10% fetal bovine serum. Viral PB1 protein was detected with anti-PB1 antibody (GeneTex GTX125923). Hoechst 33342 dye was used for nucleic acid staining.
9
Antibody Characterization for Western Blot, IP, IF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used for Western blotting, immunoprecipitation, and indirect immunofluorescence were anti-Flag M2 mouse monoclonal antibody (F3165; Sigma, USA); anti-LYAR mouse polyclonal antibody (H00055646-B01P; Abnova, China); anti-NPM1 rabbit polyclonal antibody (AP2834a; ABGENT, USA); anti-HA, -GFP, and -glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibodies (PMK013C, PKM009S, and PMK043F; PMK Bio, China); anti-Histone 3.1 polyclonal rabbit antibody (p30266; Abmart, USA); rabbit polyclonal antibodies against influenza A viral proteins PB1, PB2, PA, NP, and M1 (GTX125923, GTX125926, GTX118991, GTX125989, and GTX125928; GeneTex, USA); and Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit and Alexa Fluor 594-conjugated affinipure goat anti-mouse secondary antibodies (SA00006-2 and SA00006-3; Proteintech, USA). The small-molecule compounds used in this study were CHX (cycloheximide; 100 μg/ml; 66819; Sigma, USA) and DAPI (4′,6′-diamidino-2-phenylindole dihydrochloride; 1:1,000) (C1002; Beyotime, China).
10
Detection of Influenza A Virus Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IAV proteins were detected using rabbit polyclonal antibodies anti-PB1 (GTX125923, GeneTex), anti-PB2 (GTX125926, GeneTex), and anti-NP (GTX125989, GeneTex) diluted 1:1000 in TBSTM [tris-buffered saline (TBS)/0.1% Tween 20 (Sigma-Aldrich)/5% milk]. Cellular proteins were detected using the rabbit polyclonal antibodies anti–glyceraldehyde-3-phosphate dehydrogenase (GTX100118, GeneTex) diluted 1:4000 in TBSTM and anti-RNA Pol II (ab5131, Abcam) diluted 1:100 in TBSTM; the mouse monoclonal antibodies anti-MAVS E-3 (sc-166583, Santa Cruz Biotechnology) diluted 1:200 in TMSTM and MitoTracker [113-1] (ab92824, Abcam) diluted 1:1000 TBSTM; and the rat monoclonal antibody anti-tubulin (MCA77G, Bio-Rad) diluted 1:1000 in TBSTM. Mouse monoclonal antibody anti-FLAG M2 (F3165, Sigma-Aldrich) diluted at 1:2000 was used to detect MAVS-FLAG. Secondary antibodies IRDye 800 donkey anti-rabbit (926-32213, LI-COR), IRDye 800 goat anti-mouse (926-32210, LI-COR), IRDye 680 goat anti-mouse (926-68020, LI-COR), and IRDye 680 goat anti-rat (926-68076, LI-COR) were used to detect Western signals with a LI-COR Odyssey scanner.
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!