PA5-34663

antibody from Invitrogen Antibodies

Targeting: MTOR FLJ44809, FRAP, FRAP1, FRAP2, RAFT1, RAPT1

Provider product page for PA5-34663

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Chromatin Immunoprecipitation

Other assay

Antibody data

Antibody Data
Antigen structure
References [10]
Comments [0]
Validations
Immunocytochemistry [2]
Chromatin Immunoprecipitation [1]
Other assay [19]

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Product number: PA5-34663 - Provider product page
Provider: Invitrogen Antibodies
Product name: mTOR Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Recombinant protein fragment
Description: Recommended positive controls: 293T, A431, HeLa, HepG2, mouse brain, Flag-human mTOR transfected 293T. Predicted reactivity: Mouse (99%), Rat (100%), Zebrafish (90%), Sheep (98%). Store product as a concentrated solution. Centrifuge briefly prior to opening the vial.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Concentration: 1.45 mg/mL
Storage: Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

MTOR protein structure - PA5-34663 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 1 Increased mTORC1 activity and survival in TC T FH cells. a - c Frequency and cell number of CD69 + ( a ), CD4 + CD44 + CD62L - T EM cells ( b ), and CD4 + CXCXR5 + PD1 + Bcl6 + Foxp3 - T FH ( c ) in CD4 + T cells, with the relative distribution of T FH and Foxp3 + T FR cells in CD4 + CXCXR5 + PD1 + Bcl6 + cells. d Mean fluorescence intensity (MFI) of phospho-S6 in T N cells (CD4 + CD44 - ), T Act cells (CD4 + CD44 + ), and T FH cells (CD4 + CD44 + PD-1 hi PSGL-1 lo ). e Representative mTOR staining (green) in the GC (plain line) and T cell zone (dashed line) relative to non-GC B cells (IgD + red) and CD4 + T cells (purple). Arrows point to examples of mTOR staining. Scale bars: 200 muM for 10x and 10 muM for 20x. f Frequency and number of Ki-67 + proliferating T N , T Act , and T FH cells. g Bcl2 MFI in CD4 + CXCXR5 + PD1 + T FH and CD4 + CXCXR5 - PD1 - non-T FH cells, with representative FACS plots on the left. h Bcl2 mRNA expression in T FH and non-T FH cells as determined with a Nanostring custom panel. Spleens from 9-month-old B6 and TC mice, mean + s.e.m. of N = 3-8 mice per group compared with t tests. * P < 0.05, ** P < 0.01, and *** P < 0.001

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: mTOR antibody immunoprecipitates mTOR protein in IP experiments. IP Sample: 293T whole cell lysate/extract A : Control with 3 µg of pre-immune rabbit IgG B : Immunoprecipitation of mTOR by 3 µg of mTOR antibody (Product # PA5-34663) 5% SDS-PAGE The immunoprecipitated mTOR protein was detected by mTOR antibody (Product # PA5-34663) diluted at 1:1,000. Anti-rabbit IgG (HRP) was used as a secondary reagent.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Phosphorylated mTOR (p-mTOR), total mTOR (t-mTOR), and beta-actin expression as determined by Western blot ( a ) and phosphorylated mTOR (p-mTOR) determined by confocal microscopy ( d ) in DLD-1 and HT-29 cells treated with 5-FU (5-FU 50 uM), MM-129 (MM 1 uM, 10 uM, 100 uM), and their combination (MM 10 uM + 5-FU 50 uM) for 24 h. The samples used for electrophoresis consisted of 20 ug of protein from 6 pooled cell extracts. The samples used for electrophoresis consisted of 20 ug of protein from 6 pooled cell extracts from independent experiments ( n = 6). Band staining was quantified by densitometry ( b , c ). The corresponding uncropped blots are shown in Supplementary Figures S3a-c and S4a-c . Cells were incubated with rabbit polyclonal antibody against phospho mTOR and secondary goat polyclonal antibody against rabbit (red label). The nuclei were stained with Hoechst 33342 (blue label) ( d ). The results are presented as means +- SDs; *** p < 0.001 vs. CON, ^^^ p < 0.001 vs. 5-FU.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4. CDCA4 affected cell cycle and AKT/mTOR signaling pathway. (a-b) GSEA was utilized to identify the relative signaling pathway associated with high CDCA4 expression. (c-f) The effects of CDCA4-knockdown (c-d) and -overexpression (e-f) on the AKT/mTOR relative markers and Cyclin D1 protein were evaluated using western blot analysis. (d) Quantification of c. (f) Quantification of e. ** p < 0.01 vs . si-NC or vector group.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 1 Characterizing the CD-principal-cell targeted mTOR deletion. (A) To evaluate cellular location of Cre-recombinase activity, LacZ reporter mice were crossed with Aqp2 c re transgenic mice and the kidneys of offspring probed for beta-galactosidase activity- blue stain; left (Cre-negative); right (Cre-positive) demonstrating staining specific to renal collecting duct in Cre positive mice. mTOR CD-KO mice were generating by crossing male Mtor t m1.2K oz /J with female Aqp2 c re mice. Western blots of cortex and inner medulla homogenates (equal amounts of protein were loaded in each lane, 10 mug/lane) from wild-type (WT) and knockout (KO) (B) male and (C) female mice ( n = 6/group) probed with anti-mTOR polyclonal antibody; (D) densitometric summaries of western blotting of mTOR in cortex (CTXH) and inner medullary homogenates (IMH, mean +- sem); (E) dot blot of inner medulla (IMH), outer medulla (OMH), and cortex (CTXH) homogenates (2 mul/dot) from WT and KO male and female mice ( n = 6/group) probed with anti-mTOR antibody. (F) Ponceau S staining of proteins on dot blot for loading correction of mTOR dot blot; (G) density summary of dots normalized by Ponceau intensity. Two-way ANOVA (genotype x sex) is shown below bars; * indicates a significant ( p < 0.05) difference between WT and KO.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 2 Mechanistic Target of Rapamycin (mTOR) immunohistochemistry (IHC) in mouse kidney. Paraffin-embed kidney coronal sections were prepared for double-labeling IHC by standard chromogenic approaches; mTOR (brown) and AQP2 (gray) in male KO and WT mouse kidneys under control dietary conditions. Sections were imaged at 40x, 100x, 200x, and 600x total magnification, as indicated. At 200x, blue and orange arrows point to collecting duct and thick ascending limbs, respectively, which displayed relatively stronger mTOR labeling than other cells of the kidney (including proximal tubule). Glomerular (G) also displayed strong mTOR labeling. Under 600x, orange and lime-green arrows indicate principal and intercalated cells, respectively. Principal cells were identified by dual gray and brown label. KO mice had reduced brown labeling in cells co-staining with gray.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Double immunofluorescence staining of embryonic wild-type ( a ) and yotari ( b ) and postnatal wild-type ( c ) and yotari ( d ) mouse kidneys with the mTOR and RIP5 markers. Arrows show the expression pattern of mTOR and RIP5 in the mesenchyme (mm), renal vesicles (rv), immature glomeruli (g), convoluted tubules (Ct), ampullae (A), and collecting ducts (Cd), glomeruli (G), proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) indicated on 4',6-diamidino-2-phenylindole (DAPI) image. Immunoexpression of mTOR, RIP5, DAPI staining and merged mTOR, RIP5, and DAPI at embryonic day E15.5 in wild type ( a ) and yotari ( b ). Immunoexpression of mTOR, RIP5, DAPI staining and merged mTOR, RIP5, and DAPI at postnatal day 14 (P14) in wild type ( c ) and yotari ( d ). Wild-type and yotari mice at embryonic day E13.5 of kidney development mostly correspond regarding localization and intensity of mTOR; thus, a representative image was selected from E15.5. Wild-type and yotari mice at postnatal day 4 (P4) and 11 (P11) of kidney development mostly correspond regarding localization and intensity of mTOR; thus, a representative image was selected from P14. The most prominent protein expression area is shown in inserts, corresponding to the dashed boxes. An asterisk denotes the zone where co-expression was detected. Images were taken on magnification x40. The scale bar is 50 mum, which refers to all images. The distribution of the percentages of mTOR positive cells in the metanep

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 ACSL4 regulated the AMPK/mTOR pathway in sev-stimulated SH-SY5Y cells. SH-SY5Y cells were transfected with si- ACSL4 or si-NC for 24 h and treated with 4.1% sev for 6 h, and then tested for the expression of AMPK, pAMPK, mTOR, and p-mTOR using western blot. (A) Representative images for western blot of AMPK, pAMPK, mTOR, and p-mTOR expression in SH-SY5Y cells are shown. Quantification of (B) p-AMPK/AMPK and (C) p-mTOR/mTOR ratios in SH-SY5Y cells. **, P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Knockdown of YAP1 and mTOR inhibited the progression of bladder cancer. (A and B) RT-PCR and western blot assays were performed to test the expression of YAP1 and mTOR in bladder cancer and normal bladder tissues ( *** P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Crosstalk between the YAP1 and mTOR proteins. (A and B) HT-1376 and J82 cells were transfected with OE-mTOR and OE-NC; then cells were harvested and subjected to RT-PCR and western blot assays to determine the expression of mTOR at the mRNA and protein levels, respectively. (C-F) RT-PCR and western blot assays were performed to determine the mRNA and protein expression of YAP1 and TEAD after 48 h of HT-1376 and J82 cell transfection with OE-mTOR or OE-NC. (G) Immunofluorescence assay was performed to evaluate the effects of subcellular location of the YAP1 protein. (H) Statistical analysis of the fluorescence intensity of the YAP1 protein ( * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Upregulation of YAP1 promoted the expression and protein stability of mTOR. (A and B) HT-1376 and J82 cells were transfected with OE-YAP1 and OE-NC; then, the cells were harvested and subjected to RT-PCR and western blot assays to determine the expression of YAP1. (C and D) western blotting assays were performed to determine the protein expression and phosphorylation of mTOR, p-mTOR, p-eIF4E, eIF4E, p-rpS6 and rpS6 after HT-1376 and J82 cells were transfected with OE-YAP1 or OE-NC. (E and F) After HT-1376 and J82 cells were transfected with OE-YAP1 and OE-NC for 24 h, they were incubated with 100 ug/ml CHX for 0, 1, 2, 4, 8 and 24 h; then, cells were harvested and protein samples were extracted for western blotting with mTOR antibody. (G) Immunoprecipitation (IP) assay was performed to explore the effects of YAP1 upregulation or MG132 on mTOR expression and ubiquitination in HT-1376 and J82 cells ( * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Interaction between YAP1 protein and mTOR protein in HT-1376 and J82 cells. (A) Immunofluorescence assay was performed to evaluate the subcellular localization of the YAP1 and SKP2 proteins. (B) Duolink assay was performed to evaluate the subcellular localization of the YAP1 and mTOR proteins. (C and D) Western blotting was performed to assess the expression of flag and YAP1 after J82 and HT-1376 cells were transfected with flag-tag or YAP1-flag-tag vector ( * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Detection of the effects of YAP1/SKP2 axis on mTOR ubiquitination and bladder cancer progression. (A and B) HT-1376 and J82 cells were trans-fected with siRNAs-SKP2; then, the cells were harvested and subjected to RT-PCR and western blot assays to determine the knockdown efficiency of SKP2 ( ** P