We used 10% SDS-PAGE gels to separate equal amounts of protein and transferred the proteins onto polyvinylidene fluoride (PVDF) membranes for detection. The membranes were then sequentially incubated with specific anti-bodies at 4°C overnight; the membranes were then incubated with secondary anti-bodies at room temperature for 1 h the protein bands were detected using enhanced chemiluminescence. Anti-MTHFD1L, anti-MTHFD2 were purchased from Abcam, anti p-S6RP were purchased from Cell Signaling Technology, and anti-β-Actin was purchased from Proteintech (Wuhan, China).
Anti p s6rp
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-p-S6RP is a primary antibody that detects phosphorylation of the ribosomal protein S6 (Ser235/236). It can be used in various immunodetection techniques, such as Western blotting, to study the activation of the mTOR signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti s6rp, by Cell Signaling Technology (2 mentions)
Anti mthfd2, by Abcam (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Streptavidin biotin kit, by Nichirei Biosciences (1 mentions)
Anti p 4ebp1, by Cell Signaling Technology (1 mentions)
Anti cd31, by Cell Signaling Technology (1 mentions)
Ab190289, by Abcam (1 mentions)
Ethyl cinnamate, by Merck Group (1 mentions)
Anti ki67, by Agilent Technologies (1 mentions)
B 1325, by R&D Systems (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Alphaview software 3, by Cell Biosciences (1 mentions)
Peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Bio image analysis system, by Cell Biosciences (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Anti p70s6k, by Cell Signaling Technology (1 mentions)
Anti p p70s6k, by Cell Signaling Technology (1 mentions)
Rad001, by Selleck Chemicals (1 mentions)
Mevinolin, by Merck Group (1 mentions)
5 protocols using anti p s6rp
1
Immunoblot Analysis of Metabolic Enzymes
2
Immunohistochemical Analysis of Tumor Xenografts
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Paraffin-embedded sections of tumors from the mouse xenograft models were prepared and stained with hematoxylin and eosin (HE). To detect activation of AKT-mTOR pathway, sections were stained with anti-CD31, anti-p-S6RP, anti-p-p70S5K and anti-p-4EBP1 antibodies (Cell Signaling Technology, Danvers, MA, USA). Staining was detected using a streptavidin-biotin kit (Nichirei, Trappes, France) according to the manufacturer's protocol.
3
Kidney Organoid Fixation and Imaging
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The chip device was taken out of the incubator for fixation, and the lid was carefully removed in a sterile biosafety cabinet. The kidney organoids adherent on the surface of the ECM were rinsed with PBS, carefully overlaid with 4% PFA, and fixed for 45 min at RT. After fixation, the samples were washed three times with 0.1% Triton X-100 (Sigma-Aldrich) for 30 min. Organoids were stained and cleared with ethyl cinnamate (Sigma-Aldrich), as described before (78 (link)). The following antibodies were used in these studies: anti-CDH1 (Abcam, ab11512), anti-CFTR (Sigma-Aldrich, HPA021939), anti-LTL (Vector Laboratories, B-1325), anti-PODXL (R&D Systems, AF1658), anti-KI67 (Dako, m7248), anti-pS6RP (Cell Signaling Technology, 2211S), anti-RAC1 (Developmental Studies Hybridoma Bank, CPTC-RAC1-2), and anti-FOS (Abcam, ab190289). Samples were examined with a Zeiss TIRF/LSM 710 confocal microscope or a Leica STELLARIS 8 confocal microscope and by using Imaris 3D software (Bitplane).
4
Western Blot Analysis of S6 Ribosomal Protein
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Twenty μg protein from kidney homogenate were separated on 15% (wt/vol) poly acrylamide denaturing gels and electro-blotted onto Hybond-C membranes. These membranes were blocked with 5% (wt/vol) milk, separately probed with anti-S6RP (Cell Signaling Technology, Boston, USA) and anti-p-S6RP (Cell Signaling Technology). For the loading control, the same membranes were probed with anti-β-actin antibody (1:10,000 dilution, Abcam, Cambridge, UK), then incubated with peroxidase-conjugated secondary antibodies (1:10,000 dilution, Jackson ImmunoResearch, West Grove, USA) at room temperature for 1 h. Immunoreactive bands were visualized using ECL substrate (Thermo Fisher Scientific, Rockford, USA) and a Bio-Image Analysis System (Cell Biosciences, Inc., Santa Clara, USA). The semi-quantitative analysis results were expressed as optical volume density (OD × mm2) and normalized by β-actin for loading (AlphaView Software 3.3, Cell Biosciences, Inc.).
5
Mevinolin and Akt Pathway Modulation
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Mevinolin was from Sigma-Aldrich while MK2206 and RAD001 were purchased from Selleck Chemicals. For western blotting, primary antibodies anti-prelamin A (Sc-6214) and anti-OPG were from Santa Cruz Biotechnologies. Anti-Ser 473 p-Akt, -Thr 308 p-Akt, anti-Akt, anti-p-S6RP, anti-S6RP, anti-FLAG, anti-p-P70S6K, anti-P70S6K, anti-actin, rabbit polyclonal and mouse monoclonal were from Cell Signaling Technologies. Anti-TGFbeta 2 (ab 10850) and anti-cathepsin K antibodies were from Abcam.
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