Tuberin

The cell lysates homogenates or kidney cortex were prepared for Western blot analysis as previously described [36 (link)]. Protein concentrations were determined using the Bradford assay reagent [37 (link)]. Western blot analysis was performed as described previously [38 (link)]. Tuberin, HIF1a, p-AMPK, AMPK, tubulin, and IRS-1 antibodies were purchased from Cell Signaling (Boston, MA, USA). The GADPH antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The TP63 antibody was purchased from Thermo Scientific (Waltham, MA, USA). Protein expression was identified byusing an enhanced chemiluminescence kit (Amersham, Piscataway, NJ, USA). The expression of each protein was quantified by densitometry using the National Institutes of Health image J software (Bethesda, MD, USA) and normalized to the loading control.

Kidney homogenates were prepared as described previously [32 (link)]. Protein concentrations were determined with the Bradford assay [38 (link)] using bovine serum albumin as a standard. Western blot analysis was performed as described previously [39 (link)]. Phospho-tuberin, tuberin, phospho-p70S6K, p70S6K, phospho-Akt, Akt and Ki67, antibodies were from Cell Signaling (Beverly, MA); GAPDH antibody was obtained from Santa Cruz Biotechnology. After extensive washing of membrane with Tris-buffered saline Tween-20 buffer, anti-rabbit IgG conjugated with horseradish peroxidase was added at a 1:5000 dilution and incubated for 1 hour at room temperature. An enhanced chemiluminescence kit (Amersham, NJ) was used to identify protein expression. Expression of each protein was quantified by densitometry using National Institutes of Health image 1.62 software and normalized to a loading control.

Rapamycin (ENZO), Torin1 (cayman), AZD6244 (cayman), Parthenolide (cayman), Bortezomib (cayman), prostaglandin E2 (MCE). Antibodies used were Anti‐glutaredoxin‐1 (Abcam, catalogue ab45953); Bim (ENZO, catalogue ADI‐AAP‐330‐E); β‐actin (EASYBIO, catalogue BE0037); Anti‐Glutathione (Santa Cruz, catalogue sc‐52399); p65 (Santa Cruz, catalogue sc‐8008); Bim (Santa Cruz, catalogue sc‐374358). The antibodies below are from Cell Signaling Technology: Tuberin (catalogue 4308); phospho‐Akt (S473) (catalogue 9271); phospho‐S6(Ser235/236) (catalogue 2211); Raptor (catalogue 2280); Rictor (catalogue 2114); cleaved caspase3 (catalogue 9661); cleaved PARP (catalogue 9546); Cox2 (catalogue 12282); phospho‐p65(Ser536) (catalogue 3033); phospho‐ERK1/2(T202/Y204) (catalogue 9101).

Cells were lysed in lysis buffer (5 mM EDTA, 100 mM deoxycholic acid, 3% sodium dodecyl sulfate, Sigma-Aldrich). Samples (25 µg per lane) were boiled for 5 min and analyzed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After transfer to nitrocellulose membranes (Amersham, Arlington Height, IL, USA) and blocking at room temperature for 1 h with 5% dry milk (Merck, Darmstadt, Germany), membranes were incubated overnight at 4 °C with antibodies against MMP-2 (1:1000, Cell Signaling Technologies, Danvers, MA, USA, Cat. No. 40994), MMP-7 (1:1000; Cell Signaling Technologies, Cat. No. 71031), tuberin (1:1000; Cell Signaling Technologies, Cat. No. 3635), phospho-S6 (1:1000; Cell Signaling Technologies, Cat. No. 2211), or β-actin (1:1000; Sigma-Aldrich, Cat. No. A5441). Membranes were washed and incubated for 1 h with the appropriate secondary horseradish peroxidase conjugate antibodies (1:10,000; Thermo Scientific, Rockford, IL, USA, Cat. No. 31430/31460). The reaction was revealed by using the ECLT Prime Western Blotting System (Amersham). Images were acquired on a Kodak image station 440 CF, and densitometric analysis was performed by using Kodak 1D 3.6 Software Image (Kodak, Milano, Italy). Results were expressed as the mean value from at least three independent experiments relative to β-actin levels.