SW620 human colon cancer cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen) at 37°C and 5% CO2.15 (link) Cell lysis was performed in RIPA buffer (Thermo-Fisher, Loughborough, UK). Proteins were separated using denaturing SDS-PAGE and transferred onto nitrocellulose membranes. c-Src and phosphorylated c-Src proteins were probed with rabbit anti-Src and anti-p-Src antibodies, respectively (both from New England Biolabs, Hitchin, UK). Mouse anti-actin antibody (Abcam, Cambridge, UK) was used as a loading control. The primary antibodies were visualised with enhanced chemiluminescence reactions (ECL Prime, GE Life Sciences, Bucks, UK) using the appropriate horse radish peroxidase conjugated secondary antibodies.
Mouse anti actin antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Mouse anti-actin antibody is a primary antibody that specifically binds to actin, a highly conserved cytoskeletal protein found in all eukaryotic cells. This antibody can be used in various applications to detect and study actin expression and localization.
Automatically generated - may contain errors
Lab products found in correlation
Ecl prime, by GE Healthcare (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Hrp goat anti rabbit igg h l, by ABclonal (1 mentions)
Rabbit anti β actin, by ABclonal (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Donkey anti goat igg irdye680rd, by LI COR (1 mentions)
Donkey anti mouse igg irdye800cw secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Immobilon fl membrane, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Nupage tris acetate gel, by Thermo Fisher Scientific (1 mentions)
5 protocols using mouse anti actin antibody
1
Western Blot Analysis of c-Src Signaling
2
Western Blotting for Cellular Pathways
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For western blotting, the primary antibodies used were a mouse-anti-actin antibody, mouse anti-cytokeratin 18 antibody, and mouse anti-cytokeratin 8 antibody (Abcam, Cambridge, US), rabbit anti-caspase-3, rabbit anti-ASK1, rabbit anti-Bip/GRP78, rabbit anti-XBP1, rabbit anti-eIF2α, rabbit anti-DDIT3/CHOP, rabbit anti-FADD, rabbit anti-caspase-8, rabbit anti-FasLG, rabbit anti-Bcl-2, rabbit anti-Bax, rabbit anti-Bid, rabbit anti-Mitofusin 2, rabbit anti-AIF, rabbit anti-Cystatin C, rabbit anti-TTC11/FIS1 and rabbit anti-β-actin (ABclonal, Wuhan, China). We used secondary antibodies of HRP Goat Anti-Rabbit IgG (H + L) (ABclonal, Wuhan, China), and FITC Goat Anti Mouse IgG (H + L) antibody (AS001, ABclonal, Boston, USA). The item numbers of the used primary antibodies are depicted in Table S14 .
3
Imaging Filopodia Formation in Synovial Macrophages
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Isolated synovial macrophages were plated on MatTek cell culture dishes at low density and cultured for 2 days before the experiment. Cells were then stained using Actin (Mouse Anti-Actin antibody, abcam, Cat. # ab11003) and PSTPIP2 (Rabbit Anti-PSTPIP2 antibody, abcam, Cat. # ab155543), and secondary antibodies were used as Goat Anti-Mouse IgG(H+L) (FITC conjugated) (Elabscience, Cat. # E-AB-1015) and Goat Anti-Rabbit IgG (H+L) (AF647 conjugated) (Elabscience, Cat. # E-AB-1075). And then the plates were placed on the stage of an Olympus inverted microscope equipped with a humidified, 5% CO2-containing, 37°C temperature-controlled chamber. Phase-contrast images of five different fields were acquired at 60-s intervals using a Cooke Sensicam QE cooled CCD camera (Auburn Hills, MI) with Scanalytics IPLab software (Fairfax, VA) running on a PC. Sequences were assembled and quantified using ImageJ 1.8.0 software. Filopodia formation was assessed by visual inspection of the time-lapse movies [14 (link)].
4
Quantification of Worm Protein Levels
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4 000 L4 stage worms were pelleted by centrifugation at 1 600 rpm for 2 min and washed three times in M9 buffer containing 0.01% Tween. Afterwards, the worm pellet was re-suspended on ice in 70 μL PBS and temporarily frozen in liquid nitrogen. The extract was homogenized by sonication and centrifugation and the protein content was determined according to Bradford52 (link). The samples were diluted with sample buffer and used to quantify protein levels of hTTR and actin as control (mouse-anti actin antibody (Abcam), 1:400) by SDS-PAGE with subsequent immunoblot analysis as previously described53 (link)54 (link).
5
Quantitative Analysis of GLI3 Protein Levels
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Protein was extracted from control and INPP5E D477N/D477N organoids (derived from n=3 lines per genotype) as described previously (Magnani et al., 2010) . 20 μg protein lysates were subjected to gel electrophoresis on a 3-8% NuPAGE® Tris-Acetate gel (Life Technologies), and protein was transferred to a Immobilon-FL membrane (Millipore), which was incubated with goat anti-h/m GLI3 (1:500, R&D Systems #AF3690) and mouse anti--Actin antibody (1:15000, Abcam #ab6276). After incubating with donkey anti-goat IgG IRDye680RD (1:15000, LI-COR Biosciences) and donkey antimouse IgG IRDye800CW secondary antibodies (1:15000, Life Technologies), signal was detected using LI-COR's Odyssey Infrared Imaging System with Odyssey Software. Values for protein signal intensity were obtained using Image Studio Lite Version 4.0. GLI3 repressor and full-length protein levels and the GLI3 repressor/full length ratio were compared between control and mutant organoids using an unpaired t-test.
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