The SMO antibody was raised in rabbits (Pocono Rabbit Farm and Laboratory Inc.) using antigens and procedures described in Rohatgi et. al. [43 (link)]; and used at a dilution of 1:500. The KIF7 antibody [41 (link)] was used at 1:1000. Monoclonal antibodies against NKX2.2 and ISL1 were used at 1:10 and obtained from the Developmental Studies Hybridoma Bank. Commercially available antibodies used were: rabbit α-FOXA2 (Millipore, 1:500) rabbit α-OLIG2 (1:200, Millipore), mouse α-acetylated α-Tubulin (1:5000, Sigma Aldrich), mouse α- γ- Tubulin (1:5000, Sigma Aldrich), rabbit α-IFT88 (1:500, Proteintech), rabbit α-IFT81 (1:200, Proteintech), INPP5E (1:200, Proteintech), GPR161 (1:100, Proteintech). The GLI2 [44 (link)] and ARL13B [45 (link)] antibodies were described previously.
Inpp5e
Manufactured by Proteintech
INPP5E is a lab equipment product that functions as an inositol polyphosphate-5-phosphatase. This enzyme hydrolyzes the 5-phosphate group of various inositol phospholipids and inositol polyphosphates.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit α ift88, by Proteintech (1 mentions)
α olig2, by Merck Group (1 mentions)
Mouse α γ tubulin, by Merck Group (1 mentions)
Usp28, by Proteintech (1 mentions)
β tubulin, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by GeneTex (1 mentions)
Alexa fluor 488 and alexa fluor 555 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
4 protocols using inpp5e
1
Antibody Use for Cell Signaling Analysis
2
Comprehensive Cell Lysis and Protein Analysis
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Cell lysates were obtained in a lysis solution containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris–HCl at pH 7.5, 1 mM EGTA, 1 mM EDTA, 10% sucrose, a protease inhibitor cocktail (Roche Applied Science), and phosphatase inhibitors 0.2 mM Na3VO4 and 1 mM NaF at 4°C for 30 min. Cell or tissue lysates were separated with SDS–PAGE. Antibodies were used against p-Smad2 465/467 (1:1,000; Cell Signaling), GAPDH (1:4,000; Genetex), p53 (1:1,000; Cell Signaling or 1:500; Millipore), Dishevelled-2 (1:1,000; Cell Signaling), INPP5E (1:1,000; Proteintech), NDE1 (1:1,000; Proteintech), USP28 (1:1,000; Proteintech), 53BP1 (1:1,000; Novus Biologicals), β-tubulin (1:1,000; Santa Cruz), β-actin (1:1,000; Santa Cruz) PKD1 (E8, 1:1,000; PKD Baltimore Center), and PKD2 (G-20, 1:1,000; Santa Cruz). Densitometric quantification was performed with the Licor Image Studio software.
3
Three-Dimensional Structured Illumination Microscopy of Cell-Cell Interactions
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Three-dimensional structured illumination microscopy (3D-SIM) was preformed following the standard protocol. In brief, Jurkat cells were conjugated with SEE-loaded Raji cells at indicated time points. Cells were fixed and stained with anti-CD45 (Biotium Cat#0313) and INPP5E (Proteintech). After washing, the cells were incubated with Alexa Fluor 488- and Alexa Fluor 555-conjugated secondary antibodies (Invitrogen). The 3D-SIM images were preformed using a Zeiss ELYRA PS.1 LSM780 system equipped with a Plan Apochromat 63×/1.4NA oil-immersion objective. Z stacks with an interval of 110 nm were used to scan the whole cells. The raw images were reconstructed using ZEN software under the default parameters.
4
Immunohistochemical Analysis of Retina
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Experiments were performed as described in (56, 57) . Rats were sacrificed, and eyes were enucleated and dissected in ice-cold Ringer's solution. A retina punch (3 mm diameter) was cut from the eyecup with a surgical trephine positioned adjacent next to the optic disc, transferred onto PVDF membrane with the photoreceptor layer facing up, flat mounted between two glass slides separated by plastic spacers (ca. 240 μm) and frozen on dry ice. The retina surface was aligned with the cutting plane of a cryostat and uneven edges were trimmed away. Progressive 5-μm tangential sections were collected, subjected to SDS-PAGE and probed with antibodies against INPP5E (ProteinTech) and rhodopsin (4D2).
Measurement of ONL thickness -Mouse eye cups, with the anterior segments and lens removed, were fixed overnight in 4% PFA in PBS overnight. Eyes from both control and mutant mice were then immersed in 15% sucrose in phosphate buffer for 1 h and then to 30% sucrose overnight for cryoprotection. Twelve-micron transverse-sections were stained with DAPI and the thicknesses of the ONL layers were measured along the retinal vertical meridian at (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
approximately 500um apart on each side of the optic nerve head.
Measurement of ONL thickness -Mouse eye cups, with the anterior segments and lens removed, were fixed overnight in 4% PFA in PBS overnight. Eyes from both control and mutant mice were then immersed in 15% sucrose in phosphate buffer for 1 h and then to 30% sucrose overnight for cryoprotection. Twelve-micron transverse-sections were stained with DAPI and the thicknesses of the ONL layers were measured along the retinal vertical meridian at (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
approximately 500um apart on each side of the optic nerve head.
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