Rabbit polyclonal antibodies against the following proteins were used: CEP83 (HPA038161), CEP164 (SAB3500022) and TTBK2 (HPA018113) (Sigma); CP110 (A301-343A, Bethyl Laboratories); CEP135 (ab75005), Actin (ab8227) (Abcam); FBF1 (11531-1-AP), TCTN1 (15004-1-AP), SCLT1 (14875-1-AP), MKS1 (16206-1-AP), INPP5E (17797-1-AP) and IFT88 (13967-1-AP) (Proteintech). Rabbit antibodies against PIPKIγ, PIPKIα and PIPKIβ were described previously32 (link). Mouse antibodies against the following proteins were used: Centrin (20H5) (kindly provided by Dr Jeffrey Salisbury, Mayo Clinic); polyglutamylated tubulin (ALX-804-885-C100, Enzo Life Sciences); ODF2 (H00004957-M01, Abnova); acetylated α-tubulin (T7451), α-tubulin (T9026), γ-tubulin (T6557), FLAG (F1804) and HA (H3663) (Sigma); GFP (A-11120, Invitrogen); PI(4)P (Z-P004) and PI(4,5)P2 (Z-P045) (Echelon). Mouse monoclonal NPHP1 antibody (IgG 1) was generated at Abmart. For western blots, we used a 1:5,000 dilution for antibodies except for those against α-tubulin and actin (1:20,000). For immunofluorescence microscopy, a 1:500 dilution for antibodies was used except for those against NPHP1 (1:50), acetylated α-tubulin, polyglutamylated tubulin, centrin (1:2,000), PtdIns(4)P and PtdIns(4,5)P2 (1:100). Uncropped western blots are shown in Supplementary Fig. 7 .
Ift88 13967 1 ap
Manufactured by Proteintech
Sourced in United States
IFT88 (13967-1-AP) is a primary antibody used in research applications. It recognizes the IFT88 protein, which is a component of the intraflagellar transport complex involved in the assembly and maintenance of primary cilia. The antibody can be used for applications such as Western blotting, immunohistochemistry, and immunofluorescence.
Automatically generated - may contain errors
Lab products found in correlation
α tubulin, by Merck Group (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Alx 804 885 c100, by Enzo Life Sciences (1 mentions)
Cep135, by Abcam (1 mentions)
Rab11, by Thermo Fisher Scientific (1 mentions)
Centrin, by Merck Group (1 mentions)
Cep164, by Santa Cruz Biotechnology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Alexa fluor conjugates, by Thermo Fisher Scientific (1 mentions)
Lsm880 airyscan system, by Zeiss (1 mentions)
Arl13b 17711 1 ap, by Proteintech (1 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Lc3 nb100 2220, by Novus Biologicals (1 mentions)
Phospho ampk t172, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Cell Signaling Technology (1 mentions)
6 protocols using ift88 13967 1 ap
1
Antibody Characterization for Centrosomal Proteins
2
Immunofluorescence Imaging of Centrosomal Proteins
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Antibodies used included: Mib1 (M5948; Sigma-Aldrich, St. Louis, MO), PCM1 (H262; Santa Cruz, Santa Cruz, CA), Cep131 (A301-415A; Bethyl Laboratories, Montgomery, TX), Cep90 (Kim and Rhee, 2011 (link)), Ki-67 (ab15580; Abcam, Cambridge, MA), GT335 (AG-20B-0020-C100; Adipogen, Switzerland), BBS4 (Kim et al., 2012 (link)), Rab11 (71–5300; Life Technologies, Carlsbad, CA), Rab8 (a gift from J. Peranen, University of Helsinki, Helsinki, Finland and 610844; BD Biosciences, San Jose, CA), α-tubulin (T5168; Sigma-Aldrich), Cep290 (A301-659A; Bethyl Laboratories), Ofd1 (a gift from J. Reiter, University of California, San Francisco, USA; Singla et al., 2010 (link)), Talpid3 (Kobayashi et al., 2014 (link)), Myc (sc-40; Santa Cruz), Flag (F3165; Sigma-Aldrich), centrin (04–1624; Millipore, Billerica, MA), GFP (G1544; Santa Cruz), Cep164 (a gift from Eva Lee, University of California, USA), IFT88 (13967-1-AP; Proteintech, Chicago, IL).
3
Cryosectioning and Immunofluorescence of Chick Embryo Tissue
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Chick embryos [between embryonic day 3 (E3) to E4] were fixed in 4% formaldehyde and dehydrated in 30% sucrose overnight. These were then mounted in 1.5% Luria-Bertani (LB) agar, dehydrated in 30% sucrose again, and then snap frozen on dry ice. Cryosections of 20-μm thickness were then collected using a Leica CM3050s cryostat and then processed for immunofluorescence. For immunofluorescence, samples were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) and blocked in 1% donkey serum (Sigma-Aldrich). Primary antibodies were used at the following dilutions: Tuj1 (80120, BioLegend) 1:1000, Arl13b (17711-1-AP, Proteintech), Smo (20787-1-AP, Proteintech) 1:50, IFT88 (13967-1-AP, Proteintech) 1:200, and GPR161(13398-1-AP, Proteintech). All secondary antibodies were Alexa Fluor conjugates (Life Technologies) and used at 1:500. Images were acquired using a 60× 1.40 numerical aperture (NA) objective on a Zeiss Cell Observer Z1 microscope system (Carl Zeiss). 3D images of the primary cilium were acquired using a Zeiss LSM 880 Airyscan system equipped with a 60× 1.40 NA objective.
4
Western Blot Analysis of Cellular Proteins
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Cell lysates were prepared in 2x Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% [v/v] glycerol, 2% [w/v] SDS, 5% [v/v] β-mercaptoethanol, and 0.01% [w/v] bromophenol blue) (#161-0737, Bio-Rad, Hercules, CA). After separation in 10–12% SDS-PAGE, the proteins were transferred onto PVDF membrane (#162-0177, Bio-Rad). The membranes were then incubated with the following primary antibodies: OPA1 (#612606, BD, San Jose, CA), Drp1 (#611738, BD), ATG5 (ab54033, Abcam, Cambridge, UK), IFT88 (13967-1-AP, Proteintech, Chicago, IL), OFD1 (22851-1-AP, Proteintech), LC3 (NB100-2220, Novus Biologicals, Littleton, CO or L7543, Sigma-Aldrich), p62 (#5114, Cell Signaling Technology, Danvers, MA), AMPK (#1596, Epitomics, Burlingame, CA), phospho-AMPK (T172) (#2535, Cell Signaling Technology), cleaved caspase-3 (#9661S, Cell Signaling Technology) and actin (MAB1501, Millipore, Temecula, CA). For protein detection, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Pierce, Rockford, IL). Chemiluminescent signals were developed using Clarity Western ECL substrate (W3680-010, Bio-Rad). Densitometry was performed on scanned immunoblots using the AE-9300 Ez-Capture MG Hours Image Saver HR image capture tool (WSE-7120L, ATTO, Tokyo, Japan). Each protein expression level was normalized to that of actin.
5
Western Blot Analysis of Cell Lysates
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Cell lysates were prepared in 2× Laemmli sample buffer [62.5 mM Tris-HCl, pH 6.8, 25% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) β-mercaptoethanol, and 0.01% (w/v) bromophenol blue] (#161–0737, Bio-Rad, Hercules, CA, USA). After separation via 10–12% SDS-PAGE, the proteins were transferred onto a PVDF membrane (#162–0177, Bio-Rad). The membranes were then incubated with the following primary antibodies: Drp1 (#611738, BD), IFT88 (13967-1-AP, Proteintech, Chicago, IL, USA), HSPA9 (Sc-13967, Santa Cruz Technology, CA, USA), Gli2 (18989-1-AP, Proteintech) p-AKT (#9271S, Cell Signaling Technology), cleaved caspase-3 (#9661S, Cell Signaling Technology), and actin (MAB1501, Millipore, Temecula, CA, USA). For protein detection, the membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Pierce, Rockford, IL, USA). Chemiluminescent signals were developed using Clarity Western ECL substrate (W3680-010, Bio-Rad).
6
Immunoblotting and Immunofluorescence Protocols
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The following primary antibodies were used (western-blot WB, immunofluorescence IF, immunoprecipitation IP): IFT88 #13967-1-AP (WB: 1/500, IF: 1/250, IP: 2 µg) from Proteintech, α-tubulin (DM1α, Sigma-Aldrich #T6199, WB: 1/400), FITC-conjugated α-tubulin (DM1α, Sigma-Aldrich #F2168, IF: 1/300), NuMA (Santa-Cruz sc-365532 IF: 1:200; Abcam ab-36999 WB: 1/500) and DAPI (Cell signaling, IF: 1/10000). IgG from rabbit serum (Sigma-Aldrich I5006) were used as control IP. Secondary antibodies include for IF: Alexa Fluor 488 (#4412S or #4408S) or 555 (#4413 S or #4409S) -conjugated anti-rabbit or anti-mouse secondary antibodies (Molecular Probes, 1/1500) and for WB: anti-mouse and anti-rabbit IgG, HRP linked antibody (Cell signaling #7076 and #7074, 1/5000).
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