Each antibody was tested in at least three experimental replicates from independent cultures. Cell lysates were collected from 80% confluence starved cultures in RIPA buffer with proteinase inhibitors (SIGMA). Lysates were cleared by centrifugation and quantified (Pierce BCA protein assay kit) to equal loading. 40 ug of each lysate were loaded with Laemmli buffer and run by 10% acrylamide SDS-PAGE gels and transfer to PVDF membranes. Membranes were saturated for 1 h and antibody-incubated overnight in 5% milk in TBST. HRP secondary antibody and ECL-film exposure was used to detect bands. Antibodies used were: IFT81 (Proteintech 1:1000); CMG or IFT74 (Abcam 1:500); IFT52 (Proteintech 1:1000); IFT88 (Proteintech 1:500); Aceto-Tubulin (SIGMA T6793 1:2000); KIF3A (Sigma 1:2000) Gli3 (R&D 1:200); GAPDH (Cell Signaling 1:1000). HRP secondary antibodies were Cell Signaling (1:1000, anti-mouse and–rabbit) and Santa Cruz (1:3000, anti-goat). FIJI was used to quantify bands following Gel Analysis recommendations from ImageJ and Gassmann et al. (http://rsb.info.nih.gov/ij/docs/menus/analyze.html#gels )60 (link) and Mann-Whitney test was performed for statistic analysis using Prism software.
Kif3a
Manufactured by Merck Group
KIF3A is a laboratory equipment product offered by Merck Group. It is a motor protein that plays a crucial role in the transport of cargo within cells. The core function of KIF3A is to facilitate the movement of various cellular components, such as organelles and protein complexes, along microtubules.
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Lab products found in correlation
2 protocols using kif3a
1
Quantitative Immunoblotting of Cilia Proteins
2
Hedgehog Pathway Inhibitor 4 Protocol
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Hedgehog pathway inhibitor 4 (HPI-4), apocynin, and EGF were purchased from Sigma Aldrich (St. Louis, MO). TGFβ and recombinant mouse Shh ligand were from R&D Systems (Minneapolis, MN), and the PDGF-AA ligand was from BioVision (Farmingdale, NY). Commercially available antibodies were obtained from the following sources: Cell Signaling Technologies, Danvers, MA (Smad3, phospho–myosin light chain II [Thr-18/Ser-19], myosin light chain II, Rac1/2/3, and RhoA); Abcam, Cambridge, MA (acetylated tubulin, Kif3a, and Ki67); Sigma-Aldrich (tubulin, SMA [1A4] and nonmuscle myosin heavy chain II); Santa Cruz Biotechnology, Santa Cruz, CA (phospho–extracellular signal-regulated kinase 1/2 [ERK1/2; K23], ERK1 [E4], glyceraldehyde-3-phosphate dehydrogenase [GAPDH; 0411]); BD Transduction Laboratories, Mississauga, ON, Canada (E-cadherin); and Novus Biologicals, Littleton, CO (Nox4). Rabbit polyclonal anti-MRTF (BSAC) was described previously (Sasazuki et al., 2002 (link)). Rabbit polyclonal anti–polycystin-2 was a kind gift from the Johns Hopkins Center for Polycystic Kidney Disease (Baltimore, MD). All secondary antibodies were from Jackson ImmunoResearch Laboratories (Westgrove, PA).
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