Cells, grown on coverslips were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 minutes at room temperature. After permeabilization with 0.01% Triton X-100 (Sigma-Aldrich) for 5 minutes, non-specific protein binding was blocked by 10% goat serum (Sigma-Aldrich) dissolved in 2% BSA-PBS. Antibody against alpha-tubulin (1∶2000 dilution, mouse, T-6074, Sigma-Aldrich) and coumarin- or Alexa594-coupled phalloidin (1∶250 dilution, Molecular Probes) were used to label microtubules and actin filaments. Alexa Fluor 488 goat anti-rabbit (1∶400 dilution, Molecular Probes) was applied as secondary antibody. Alternatively, cells were fixed with 100% −20°C methanol (Merck) for 8 minutes, and stained with dynein (1∶100 dilution, abcam, ab23905) and alpha-tubulin (1∶1000 dilution, abcam, ab18251) antibodies.
Ab23905
Manufactured by Abcam
Sourced in United Kingdom
Ab23905 is a laboratory product manufactured by Abcam. It serves as a tool for scientific research, but a detailed description of its core function cannot be provided while maintaining an unbiased and strictly factual approach. The intended use of this product is not specified.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Ab18251, by Abcam (1 mentions)
T6074, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
A2066, by Merck Group (1 mentions)
Ab4448, by Abcam (1 mentions)
T5168, by Merck Group (1 mentions)
A11122, by Thermo Fisher Scientific (1 mentions)
S5768, by Merck Group (1 mentions)
Mab374, by Merck Group (1 mentions)
Ab108319, by Abcam (1 mentions)
Ab84571, by Abcam (1 mentions)
Las af software version 1.6.0 build 1016, by Leica (1 mentions)
T6074, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica (1 mentions)
7 protocols using ab23905
1
Immunofluorescence Staining of Cytoskeletal Structures
2
Antibody Sources for Cell Biology
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Rabbit polyclonal antibodies to human SUN1 and SUN2 have been previously described (Turgay et al., 2010 (link); Sosa et al., 2012 (link)). Antibodies against GFP and human full-length NudE were generated in rabbits and affinity purified. The mouse monoclonal antibodies against α-tubulin (T5168) and β-actin (A1978) were purchased from Sigma-Aldrich. The rabbit polyclonal antibodies against BICD2 (ab73286), pericentrin (ab4448), and NudEL (ab25959) were purchased from Abcam. Rabbit polyclonal anti–β-actin (A2066) was obtained from Sigma-Aldrich, and mouse monoclonal anti–dynein intermediate chain (DIC; ab23905) was obtained from Abcam.
3
Antibody Labeling and Protein Detection
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Antibodies used included anti-GFP (A11122, 1:1000, Invitrogen) to label transfected cells and neuronal morphologic features. The antibodies used for Western blot analyses include: anti-BDNF (ab108319, 1:3000, Abcam), anti-FLAG M2-peroxidase conjugated (1:20,000, Sigma), anti-GFP-peroxidase conjugated (clone 3D8A1B8, 1:10,000, Abking), anti-MBNL1 polyclonal antibody (ABE241, 1:1000, Millipore) [21 (link)], anti-DIC (dynein intermediate chain)(ab23905, 1:1000, Abcam), anti-DYNLL2 (Merck, ABT140), anti-PSD95 (post-synaptic density 95, clone 3H4.3, 1:2000, Millipore), anti-synaptophysin (S5768, 1:1000, Sigma), anti-TrkB (#4603, 1:1000, Cell Signaling), anti-phospho-TrkB (Y817) (MA5-32,207, 1:1000, Invitrogen), anti-HSP90 (heat shock protein 90, a gift from Dr. Yi-Ping Hsueh, 1:2000) and anti-GAPDH (MAB374, 1:20,000, Millipore). Anti-GFP antibodies (clones 19C8 and 19F7) used in immunoprecipitation were from Memorial Sloan-Kettering Monoclonal Antibody Facility. To detect the level of BDNF, the nitrocellulose membrane underwent antigen retrieval to boost the signal [22 (link)].
4
Immunohistochemical Scoring of GPSM2 and DYNC1I1
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IHC staining was performed as described previously (19 (link)). Anti-human GPSM2 rabbit antibody was used at a dilution of 1:100 (ab84571; Abcam, Cambridge, UK); Anti-human DYNC1I1 mouse antibody was used at a dilution of 1:100 (ab23905; Abcam, Cambridge, UK); phosphate-buffered saline was used as a negative control. Each section was evaluated and scored independently by two pathologists. A semi-quantitative scoring system was used in this assay. Intensity was scored as “0” (negative), “1” (weak), “2” (moderate), and “3” (strong), and the percentages of tumor cells within each category were calculated. The percentage score was multiplied by the staining intensity score to generate the IHC score. The histological score range was from zero (minimum) to 300 (maximum). Positive expression was defined as detectable immunoreactions with an IHC score > 10.
5
Immunostaining of Cell Lines
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A549, H1650, H460, Calu-6, PC9 or AALE cells were plated onto poly-D -lysine-coated eight-well glass chamber slides (5,000 cells per well) for immunostaining. The cells were fixed with 10% buffered formalin and indirect immunofluorescence was performed as described56 (link). Primary antibodies used were phospho-TBK1 (Cell signaling #5483S) or CEP170 mouse monoclonal-72-413-1 (Invitrogen #41-3200) at 1:100 dilution or 1:500 dilution of rabbit monoclonal NuMA (EP3976 Abcam #109262), Alpha tubulin (1:2,000, Sigma #T6074), Dynein intermediate chain (Abcam#ab23905) and Kif2b (Abcam #ab98214 or Novus #NBP86002), γ Tubulin (Santa Cruz #sc51715) were used at 1:100 dilution. Anti-rabbit Alexa Fluor-488 or anti- mouse Alexa Fluor-594 (Molecular Probes) was used as secondary antibody. DAPI (Vector Labs) was used to stain the nuclei. Cells were visualized with a DM16000 inverted Leica TCS SP5 tandem scanning confocal microscope with a × 63/1.40NA oil immersion objective. Images and Z-stacks were produced with three cooled photomultiplier detectors and analysed with the LAS AF software version 1.6.0 build 1016 (Leica Microsystems, Germany).
6
Colocalization of Kv7.4, Caveolin-1, and Dynein
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Colocalization of dynein with Kv7.4 or caveolin-1 and caveolin-1 with Kv7.4 was studied with PLA in HEK293B cells stably expressing Kv7.4 and Kv7.4-Q580A or freshly isolated rat mesenteric artery myocytes using the Duolink in situ (PLA) detection kit 563 (Olink) per the manufacturer’s instructions. Similar to previous studies (Zhong et al., 2010a (link); Brueggemann et al., 2014 (link); Chadha et al., 2014 (link); Jepps et al., 2015 (link); Stott et al., 2016 (link); Barrese et al., 2020 (link)), cells were allowed to adhere to coverslips and fixed in 4% paraformaldehyde in PBS. Cells were permeabilized in PBST, blocked in Duolink blocking solution, and incubated with pairs of primary antibodies. The primary antibodies employed were dynein (ab23905; Abcam), Kv7.4 (ab65797; Abcam), caveolin-1 (1:500 ab17052; Abcam), caveolin-1 (C3237; Sigma-Aldrich), and NCX (R3F1; Swant). Combinations of secondary anti-rabbit or anti-mouse antibodies of PLA PLUS and MINUS probes were used followed by hybridization, ligation, and amplification steps. Colocalization signals (proteins located within 40 nm of each other) were visualized with a standard Zeiss LSM710 upright laser-scanning confocal microscope.
7
Comprehensive Cell Culture Immunoassays
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Cell culture reagents, including Lipofectamine 2000 that was used for transfection, were purchased from Thermo Fisher Scientific (Waltham, MA). Antibodies against DCX (ab18723), DHC (ab6305), GM130 (ab169276, ab52649), JIP3 (ab196761), and DIC (ab23905) were purchased from Abcam (Cambridge, MA). Antibodies against CLASP2 were obtained from Santa Cruz (sc-376496, Dallas, TX) or Novus Biologicals (NBP1-21394, Centennial, CO). Antibodies against HA were obtained from EMD Millipore (Billerica, MA).
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