P rictor

Manufactured by Cell Signaling Technology

Sourced in United States

P-Rictor is a phosphorylated form of the Rictor protein, a key component of the mTORC2 complex. mTORC2 is an important regulator of cell growth, proliferation, and survival. P-Rictor is frequently used as a marker to study the activation and function of the mTORC2 pathway.

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5 protocols using p rictor

Investigating Estrogen Receptor Signaling

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Western blot analysis used specific antibodies for ERα (HC-20, Santa Cruz); ERK2 (D-2, Santa Cruz); and pS6, pS6K, pmTOR, pRAPTOR, pRICTOR, pMAPK (Cell Signaling). Coimmunoprecipitation assays used antibodies for SRC3 (Santa Cruz, C-20) and ERα (Santa Cruz, F10). ChIP assays were carried out as described (9 (link), 11 (link)). Antibodies used were for ERα (HC20), ERK2 (Santa Cruz, D2 and C14), and pSer⁵ RNA Pol II (Santa Cruz, sc-47701). ChIP DNA was isolated using QIAGEN PCR purification kit and used for ChIP-seq analysis and quantitative real-time PCR (qPCR). qPCR was used to calculate recruitment to the regions studied, as described (9 (link)).

Madak-Erdogan Z., Kim S.H., Gong P., Zhao Y.C., Zhang H., Chambliss K.L., Carlson K.E., Mayne C.G., Shaul P.W., Korach K.S., Katzenellenbogen J.A, & Katzenellenbogen B.S. (2016). Design of pathway-preferential estrogens that provide beneficial metabolic and vascular effects without stimulating reproductive tissues. Science signaling, 9(429), ra53.

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Western Blot Analysis of Retinal Autophagy

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Retinal samples were collected, homogenized, and then centrifuged at 15,000 × g for 15 min at 4 °C. Western blotting was used to measure the levels of LC3 (Santa Cruz Biotechnology, Inc., USA), LAMP1 (Abcam, Cambridge, MA, USA), p-mTOR S2448 (Cell Signaling, Beverly, MA, USA), p-S6K (Cell Signaling, Beverly, MA, USA), p-Rictor (Cell Signaling, Beverly, MA, USA), p-Akt S473 (Cell Signaling, Beverly, MA, USA), and p-GSK3b S9 (Cell Signaling, Beverly, MA, USA) in retinal samples. Retinal protein extracts were separated using a 4–12% NuPAGE Bis-Tris gel (Invitrogen, Carlsbad, CA, USA). The separated proteins were transferred onto polyvinylidene difluoride membranes and blocked with 5% milk in Tris-buffered saline/Tween-20 containing 20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, and 0.5% Tween-20. The membranes were then blotted with mouse anti-LC antibodies, mouse anti-LAMP1 antibody, mouse anti-p62 antibody, mouse anti-mTOR antibody, and goat anti-mouse immunoglobulin (Abcam, Cambridge, MA, USA). The blots were then developed with Enhanced Chemiluminescent Substrates (Perkin-Elmer Life Science, Boston, MA, USA), and the relative intensities of the bands were measured using an image analysis system (Amersham Biosciences Uppsala, Sweden).

Wen Y.T., Zhang J.R., Kapupara K, & Tsai R.K. (2019). mTORC2 activation protects retinal ganglion cells via Akt signaling after autophagy induction in traumatic optic nerve injury. Experimental & Molecular Medicine, 51(8), 96.

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Characterization of Signaling Pathways in Human Myeloma

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Human myeloma IgE (huIgE, Calbiochem, La Jolla, CA) was biotinylated in the NIAID Core Facility. The protein-specific antibodies used in confocal microscopy were: mTOR (10343, IBL, Minneapolis, MN) and rictor (NB100-612, Novus Biologicals, Littleton, CO). The β-actin-specific antibody for immunoblotting was from Cell Signaling Technology (Beverley, MA) and Lyn-specific antibody for immunoblotting from Santa Cruz Biotechnology (Santa Cruz, CA). The conjugated protein-specific antibodies were: fluorophore-conjugated antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), horseradish peroxidase-conjugated rabbit IgG (Amersham Biosciences, Piscataway, NJ), and mouse IgG Fc-specific antibody (Sigma-Aldrich). The phosphoprotein-specific antibodies for immunoblotting were: p-mTOR(Ser2448), p-mTOR(Ser2481), p-rictor (Thr1135), p-Akt(Thr308), p-Akt(Ser473), p-LAT(Tyr171), p-S6 ribosomal protein (S6RP) (Ser240/244), p-4E-BP1(Thr37/46), and rictor (Cell Signaling Technology), and p-PLCγ₁(Tyr783) (Invitrogen). The blocking donkey serum was from Santa Cruz Biotechnology and Torin1 (37 (link)) was a gift of Dr. Nathanael S. Gray, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA. FITC-conjugated phalloidin and other chemicals and reagents were from Sigma-Aldrich.

Smrz D., Cruse G., Beaven M.A., Kirshenbaum A., Metcalfe D.D, & Gilfillan A.M. (2014). Rictor negatively regulates FcεRI-induced mast cell degranulation. Journal of immunology (Baltimore, Md. : 1950), 193(12), 5924-5932.

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Rapamycin Modulates mTOR Signaling

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To examine the effects of rapamycin, Ad-hMSCs were incubated for 48 h in the presence of rapamycin. The membrane was incubated overnight at 4 °C with primary antibodies against the following: p-mTOR, mTOR, p-Rictor, Rictor, p-Raptor, Raptor, Beclin1, ATG5, ATG7, LC3 II (all antibodies were from Cell Signaling Technology Inc., Danvers, MA, USA), and β-actin (Sigma, Sigma-Aldrich, St Louis, MO). After washing in Tris-buffered saline and Tween 20 (TTBS), the reactive bands were visualized using an ECL detection kit and Hyperfilm-ECL reagents (Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK).

Kim K.W., Moon S.J., Park M.J., Kim B.M., Kim E.K., Lee S.H., Lee E.J., Chung B.H., Yang C.W, & Cho M.L. (2015). Optimization of adipose tissue-derived mesenchymal stem cells by rapamycin in a murine model of acute graft-versus-host disease. Stem Cell Research & Therapy, 6, 202.

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Profiling Cellular Signaling Pathways

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All the cell culture reagents were from HyClone (Logan, UT, USA). Antibodies against Raptor (#2280S), p-Raptor (#2083S), p70S6K1 (#2708S), p-p70S6K1 (#9206S), p-4E-BP1 (#13443S), Rictor (#2114), p-Rictor (#3806S), p-GSK3β (#9336L), p-S6 (#4858S), N-cadherin (#14215S), Slug (#9585S), caspase 3 (#14220S), p21 (#2947), CDK4 (#12,790), CDK6 (#13,331), p-Rb (#9308S) were from Cell Signaling Technology. Antibodies against GSK3β (22104-1-AP) and caspase 9 (10380-1-AP) were obtained from Proteintech. Antibodies specific for S6 (sc-74,459), vimentin (sc-32,322), PARP-1 (sc-8007), Rb (sc-74,562) and Cyclin D1 (sc-20,044) were procured from Santa cruz biotechnology. Antibody against β-actin (A1978) was supplied by Sigma. Z86 was obtained as previously reported [13 (link)].

Nie S., Chang L., Huang Y., Zhou H., Yang Q., Kong L, & Li Y. (2024). β-carboline derivative Z86 attenuates colorectal cancer cell proliferation and migration by directly targeting PI3K. Natural Products and Bioprospecting, 14(1), 3.

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