Western blot samples from gastric cancer cell lines were collected and processed as described elsewhere [18 (link)]. The rabbit anti-human FHOD1 or FMNL1 (1:1000, Sigma-Aldrich, St. Louis, MO) antibody was incubated overnight at 4 °C. Rabbit polyclonal to GAPDH—HRP conjugated (Abcam, Cambridge, UK) was used 1:5000 as a control for protein loading. The secondary antibodies were HRP-conjugated swine anti-rabbit and HRP-conjugated rabbit anti-mouse immunoglobulins (1:3000, Dako, Glostrup, Denmark). Membranes were washed three times with TBST between the different steps.
Fhod1
Manufactured by Merck Group
Sourced in United States
FHOD1 is a laboratory equipment product offered by Merck Group. It is a specialized device used for scientific research and analysis, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Further information may be available from Merck Group directly.
Automatically generated - may contain errors
Lab products found in correlation
Daam1, by Proteintech (3 mentions)
Hrp conjugated rabbit anti mouse immunoglobulins, by Agilent Technologies (1 mentions)
Nanozoomer s60 slide scanner, by Hamamatsu Photonics (1 mentions)
Ndp view2 image viewing software, by Hamamatsu Photonics (1 mentions)
Rabbit anti erk2 antibody, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti p44 42 mapk erk1 2 antibody, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 or 568 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Eclipse ni fluorescence microscope, by Nikon (1 mentions)
Gelatin, by Merck Group (1 mentions)
Alexa fluor 568 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mounting with dapi, by Thermo Fisher Scientific (1 mentions)
4 protocols using fhod1
1
Western Blot Analysis of FHOD1 and FMNL1 in Gastric Cancer
2
Immunostaining and Scoring of FHOD1, INF2, and DAAM1
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TMAs were sectioned at 3.5 μm and stained with rabbit anti-human FHOD1 (1:500; Sigma-Aldrich, USA), INF2 (1:500; Proteintech, USA), and DAAM1 (1:200; Proteintech). Immunostaining was performed according to the streptavidin-peroxidase method, using a Labvision staining device (Thermo Fisher Scientific, USA). Stained slides were scanned using a NanoZoomer S60 slide scanner (Hamamatsu Photonics, Japan), and a pathologist (MG) and researcher (MP) evaluated the staining intensity using the NDP.view2 Image viewing software (Hamamatsu).
3
Verifying siRNA and Pathway Inhibition
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Western blot samples were collected and processed as previously described [9 (link)]. To verify the efficacy of the siRNA treatments, we used rabbit anti-HER2/ERBB2 (Proteintech), FHOD1 (Sigma-Aldrich), INF2, and DAAM1 (both Proteintech) antibodies. PI3K/Akt pathway inhibition was verified by immunoblotting using rabbit anti-pAkt and Akt antibodies (Cell Signaling Technology), and MEK/ERK pathway inhibition was verified by immunoblotting with a rabbit anti-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology) and rabbit anti-ERK1 mixed with rabbit anti-ERK2 antibody (both from Santa Cruz Biotechnology, USA). All primary antibodies were used at 1:1000 dilution. The secondary antibodies used were horseradish peroxidase-conjugated swine anti-rabbit or rabbit anti-mouse (Agilent, USA) diluted 1:3,000 in blocking solution. The control for protein loading was CoraLite® Plus 488-conjugated glyceraldehyde 3-phosphate dehydrogenase mouse monoclonal antibody (Proteintech) at 1:5,000.
4
Immunostaining of Actin-related Proteins
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Cells were seeded onto 13 mm coverslips coated with 100 µg/mL gelatin (Sigma-Aldrich) and cultured in complete medium for 24 hours. Staining was performed as previously described [9 (link)]. The primary antibodies used were rabbit anti-human HER2/ErbB2 (Proteintech), FHOD1 (Sigma-Aldrich), INF2 (Proteintech), and DAAM1 (Proteintech) (all diluted 1:100). The secondary antibody was Alexa Fluor 568 goat anti-rabbit IgG (1:500; Invitrogen, USA). For the visualization of actin filaments, Alexa Fluor 488- or 568-conjugated phalloidin (1:500; Invitrogen) was incubated alone or together with the secondary antibody. The mounting medium contained 4′,6-diamidino-2-phenylindole (DAPI) for staining nuclei (ProLong® Gold Antifade Mounting with DAPI; Thermo Fisher Scientific). Images were captured using a Nikon Eclipse Ni fluorescence microscope (Nikon, Japan), and different channels were merged using the ImageJ 1.53t software (http://rsbweb.nih.gov/ij/ ).
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