anti-ps6k (thr389)  (Cell Signaling Technology Inc)

Journal: PLOS Pathogens

Article Title: Interleukin-17A signaling promotes CD8 + T cell cytotoxicity against West Nile virus infection through enhancing PI3K-mTOR-mediated metabolism

doi: 10.1371/journal.ppat.1013218

Figure Lengend Snippet: (A) Eight weeks old Il17ra -/- , and WT (C57BL/6J) mice (n = 3) were infected with 100 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i., followed by purification of CD8 + T cells. The transcription profile was analyzed by RNA sequencing and the representative data were shown as a heatmap. (B-D) Eight to nine weeks old Il17a -/- (n = 5), Il17rc -/- (n = 4), and WT (n = 8) mice were infected with 20 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i. CD8 + T cells were isolated from the splenocytes, cultured ex vivo with mouse rIL-17A (50 ng/ml) or RPMI (control) for 24 h and measured the gene expression of PIK3ca (B), mTOR (C), and S6K1 (D) by RT-qPCR, expressed as the relative fold changes (RFC). Data were analyzed by two-tailed student’s t- tests (B-D) and presented as mean ± s.e.m. with *, and ** denoting p <0.05, and p <0.01, respectively. (E-J) Eight to nine weeks-old WT mice were infected with 20 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i. CD8 + T cells were isolated from the splenocytes, stimulated ex vivo with mouse rIL-17A (100 ng/ml) or RPMI (control), and co-treated with either rapamycin (20 nM), LY294002 (12.5 µM) for 15- or 60- minutes. Cell lysates were resolved with SDS-PAGE. The protein expressions of PI3K, p -PI3K Tyr458 , AKT, p -AKT Thr308 , mTOR, p -mTOR Ser2481 , pS6K, p -pS6K Thr389 , 4EBP1, and p-4EBP1 Thr37/46 were analyzed by immunoblot. GAPDH was used as the loading control. (E) The protein bands of PI3K, p -PI3K Tyr458 , AKT, p -AKT Thr308 , mTOR, and p -mTOR Ser2481 were from the 15-min treatment, and the remaining bands were from the 60-min treatment. (F-J) Densitometric quantification of the phosphorylated protein normalized to corresponding total protein and analyzed by Image Lab software.

Article Snippet: The proteins were then transferred into a nitrocellulose membrane and probed with primary antibodies against mouse PI3K (p85, 1:500, Cell Signaling Technology, 4257), phosphorated ( p )-PI3K (p85 [Tyr458]/p55 [Tyr199], 1:200, Cell Signaling Technology, 4228), AKT (1:500, Cell Signaling Technology, 9272), p -AKT (Thr308, 1:200, Cell Signaling Technology, 4056), mTOR (1:500, Cell Signaling Technology, 2972), p -mTOR (Ser2481, 1:200, Cell Signaling Technology, 2974), pS6K (1:500, Cell Signaling Technology, 9202), p -pS6K (Thr389, 1:200, Cell Signaling Technology, 9234), 4EBP1 (1:500, Cell Signaling Technology, 9644), p -4EBP1 (Thr37/46, 1:500, Cell Signaling Technology, 2855), and GAPDH as control (1:1000, Cell Signaling Technology, 5174) and incubated overnight at 4°C.

Techniques: Infection, Purification, RNA Sequencing, Isolation, Cell Culture, Ex Vivo, Control, Gene Expression, Quantitative RT-PCR, Two Tailed Test, SDS Page, Western Blot, Software

Journal: PLOS Pathogens

Article Title: Interleukin-17A signaling promotes CD8 + T cell cytotoxicity against West Nile virus infection through enhancing PI3K-mTOR-mediated metabolism

doi: 10.1371/journal.ppat.1013218

Figure Lengend Snippet: (A) Eight weeks old Il17ra -/- , and WT (C57BL/6J) mice (n = 3) were infected with 100 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i., followed by purification of CD8 + T cells. The transcription profile was analyzed by RNA sequencing and the representative data were shown as a heatmap. (B-D) Eight to nine weeks old Il17a -/- (n = 5), Il17rc -/- (n = 4), and WT (n = 8) mice were infected with 20 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i. CD8 + T cells were isolated from the splenocytes, cultured ex vivo with mouse rIL-17A (50 ng/ml) or RPMI (control) for 24 h and measured the gene expression of PIK3ca (B), mTOR (C), and S6K1 (D) by RT-qPCR, expressed as the relative fold changes (RFC). Data were analyzed by two-tailed student’s t- tests (B-D) and presented as mean ± s.e.m. with *, and ** denoting p <0.05, and p <0.01, respectively. (E-J) Eight to nine weeks-old WT mice were infected with 20 PFU of WNV via f.p. inoculation, and the spleens were collected on D8 p.i. CD8 + T cells were isolated from the splenocytes, stimulated ex vivo with mouse rIL-17A (100 ng/ml) or RPMI (control), and co-treated with either rapamycin (20 nM), LY294002 (12.5 µM) for 15- or 60- minutes. Cell lysates were resolved with SDS-PAGE. The protein expressions of PI3K, p -PI3K Tyr458 , AKT, p -AKT Thr308 , mTOR, p -mTOR Ser2481 , pS6K, p -pS6K Thr389 , 4EBP1, and p-4EBP1 Thr37/46 were analyzed by immunoblot. GAPDH was used as the loading control. (E) The protein bands of PI3K, p -PI3K Tyr458 , AKT, p -AKT Thr308 , mTOR, and p -mTOR Ser2481 were from the 15-min treatment, and the remaining bands were from the 60-min treatment. (F-J) Densitometric quantification of the phosphorylated protein normalized to corresponding total protein and analyzed by Image Lab software.

Article Snippet: The proteins were then transferred into a nitrocellulose membrane and probed with primary antibodies against mouse PI3K (p85, 1:500, Cell Signaling Technology, 4257), phosphorated ( p )-PI3K (p85 [Tyr458]/p55 [Tyr199], 1:200, Cell Signaling Technology, 4228), AKT (1:500, Cell Signaling Technology, 9272), p -AKT (Thr308, 1:200, Cell Signaling Technology, 4056), mTOR (1:500, Cell Signaling Technology, 2972), p -mTOR (Ser2481, 1:200, Cell Signaling Technology, 2974), pS6K (1:500, Cell Signaling Technology, 9202), p -pS6K (Thr389, 1:200, Cell Signaling Technology, 9234), 4EBP1 (1:500, Cell Signaling Technology, 9644), p -4EBP1 (Thr37/46, 1:500, Cell Signaling Technology, 2855), and GAPDH as control (1:1000, Cell Signaling Technology, 5174) and incubated overnight at 4°C.

Techniques: Infection, Purification, RNA Sequencing, Isolation, Cell Culture, Ex Vivo, Control, Gene Expression, Quantitative RT-PCR, Two Tailed Test, SDS Page, Western Blot, Software

Journal: Aging Cell

Article Title: Activated mTOR Signaling in the RPE Drives EMT , Autophagy, and Metabolic Disruption, Resulting in AMD ‐Like Pathology in Mice

doi: 10.1111/acel.70018

Figure Lengend Snippet: Generation and characterization of mLST8 KI mice. (a) Cartoon showing that in addition to mTOR, mLST8 is a major component of both mTOR complexes 1 and 2. (b) Schematic showing the strategy for generating mLST8 KI mice. Briefly, TGA stop codon in the mouse Best1 gene was replaced with the “T2A‐mouse mLST8 CDS (coding sequence)” cassette. The targeting vector was generated by PCR using BAC clone RP23‐340G9 from the C57BL/6 library as a template. The targeting vector had Neo cassette, which was flanked by SDA (self‐deletion anchor) sites. DTA (diptheria toxin A) was used for negative selection. C57BL/6 embryonic stem cells were used for gene targeting. (c) Western blot analysis indicating that RPE lysates shows overexpression of mLST8 relative to WT. Such changes were not seen in retina lysates from the same mice. Retina RPE lysate preparation was confirmed by evaluating the levels of GFAP and RPE65 in the respective lysates n = 3. ** p < 0.01. (d) Western blot from RPE lysates of mLST8 KI RPE cells further revealed an increased ratio of p‐S6K/S6K, p‐4EBP/4EBP, p‐ULK1/ULK1, and p‐Akt1/Akt1 in these cells, compared to controls (WT) n = 3. * p < 0.05. (e) Western blot showing that mLST8 overexpression (AAV2‐ mLST8 ) in Raptor or Rictor KO MEF cells activates mTORC2 target (p‐Akt1) and mTORC1 target (p‐P70S6K), respectively, compared to controls n = 3. **** p < 0.001, ns = not significant.

Article Snippet: The cells from all the experimental groups were lysed in the CHAPS lysis buffer, and Raptor and Rictor antibodies were used to immunoprecipitate mTOR bound complexes using protein Ig beads (Thermo Fisher, 88802). mTORC1 and mTORC2 target proteins S6K1 (Sino Biological, 10099‐H09B‐50) and Akt1 (EMD Milipore, 14–279) were given to the immune‐precipitated complexes and incubated for 15 min in 37°C.

Techniques: Sequencing, Plasmid Preparation, Generated, Selection, Western Blot, Over Expression

Journal: Aging Cell

Article Title: Activated mTOR Signaling in the RPE Drives EMT , Autophagy, and Metabolic Disruption, Resulting in AMD ‐Like Pathology in Mice

doi: 10.1111/acel.70018

Figure Lengend Snippet: βA3/A1‐crystallin overexpression in the RPE rescues autophagy and melanosome alterations and retinal structure/function in mLST8 KI mice. (a) Cartoon showing the strategy for subretinal injection of the AAV2‐m Cryba1 construct into one eye of 8‐month‐old mLST8 KI mice, with the contralateral eyes receiving PBS vehicle. Animals were euthanized 2 and 4 months after injection. (b, c) Western blot analysis and densitometry showing that Cryba1 overexpression in the RPE (confirmed by western blot in b) of mLST8 KI mice could rescue the abnormal levels of both mTORC1 (p‐S6K) and mTORC2 (p‐Akt1) targets (b), and the melanosome marker (PMEL; b), as well as rescued the levels of major regulators of autophagosome formation (Atg9b, Atg7; c) in these animals (b, c), relative to PBS injected eyes (b, c). n = 3. * p < 0.05, ** p < 0.01. AAV2‐m Cryba1 treatment (subretinal injection) to mLST8 KI mice for 4 months rescued retinal function as evident from increase in scotopic (d) a‐ and (e) b‐wave amplitudes after the treatment, compared to PBS‐injected contralateral eyes of the KI mouse. n = 4. **** p < 0.0001, * p < 0.05. (f) AAV2‐m Cryba1 treatment to mLST8 KI mice for 4 months also rescued early RPE changes (arrows) like the patchy appearance of the monolayer and decline in thickness (spider plot), compared to PBS–treated contralateral eyes of the mLST8 KI mouse. n = 4. Scale bar = 20 μm. * p < 0.05. (g) Cartoon depicting overexpression of mLST8 in RPE cells ( mLST8 KI mice) activated both mTORC1 and mTORC2, disrupting glucose metabolism, mitochondrial function, autophagy, and melanosome function, leading to debris accumulation, EMT activation, and age‐related retinal degeneration resembling AMD. Targeting mTOR with inhibitors or modulators rescued these changes, suggesting a potential therapeutic strategy for retinal diseases by modulating mTOR signaling. Created with BioRender.com .

Article Snippet: The cells from all the experimental groups were lysed in the CHAPS lysis buffer, and Raptor and Rictor antibodies were used to immunoprecipitate mTOR bound complexes using protein Ig beads (Thermo Fisher, 88802). mTORC1 and mTORC2 target proteins S6K1 (Sino Biological, 10099‐H09B‐50) and Akt1 (EMD Milipore, 14–279) were given to the immune‐precipitated complexes and incubated for 15 min in 37°C.

Techniques: Over Expression, Injection, Construct, Western Blot, Marker, Activation Assay