Mouse anti β actin clone ac 15

Human recombinant TGF-β1 and AG1478 were from Calbiochem (La Jolla, CA, USA). EGF was kindly gifted by Serono Lab (Madrid, Spain). Human recombinant HB-EGF, human recombinant TGF-α, MβC, filipin III from Streptomyces filipinensis, nystatin and water-soluble cholesterol were from Sigma-Aldrich (St. Louis, MO, USA). The antibodies used were: mouse anti-β-actin (clone AC-15) from Sigma-Aldrich, rabbit anti-phospho-Akt (Ser473) (D9E) XP, rabbit anti-Akt, rabbit anti-phospho-EGFR (Tyr1068) (D7A5) XP, rabbit anti-EGFR, rabbit anti-phospho-p44/42 MAPK (Thr202/Tyr204), rabbit anti-p44/42 MAPK were from Cell Signaling Technology (Beverly, MA, USA), mouse anti-Caveolin-1 from BD Biosciences (Franklin Lakes, NJ, USA), rabbit anti-Ki67 from AbCam (Cambridge, UK), rabbit anti-TACE/ADAM17 (807-823) from Calbiochem, rabbit anti-NFκB p65, rabbit anti-TβRI (H-100, used in immunocytochemistry), rabbit anti-TβRI (R-20, used in western blot) and rabbit anti-TβRII (C-16) from Santa Cruz Biotechnologies (Dallas, TX, USA). Secondary antibodies: Alexa Fluor 488-conjugated anti-rabbit and anti-mouse from Molecular Probes (Eugene, OR, USA) and ECL Mouse IgG, and Rabbit IgG, HRP-Linked antibodies from GE Healthcare (Buckinghamshire, UK). GM1 was detected with horseradish peroxidase-tagged cholera toxin B subunit from Sigma (St. Louis, MO, USA).

One hundred microliters of RIPA lysate was added to 20 mg of tissue, homogenized on ice, centrifuged at 12,000 rev/minute for 10 minutes at 4°C, and the supernatant was separated to determine the protein concentration. The protein was separated by 10% SDS-PAGE electrophoresis with a loading of 40 µg in a volume of 20 µL. After the electrophoresis, a “sandwich” was prepared, and the protein was electrotransferred to the PVDF membrane. After blocking with 5% nonfat dry milk for 2 hours, added anti-uPA (1:600), anti-p38MAPK (1:500), and mouse anti-β-actin (clone AC-15; 1:10,000, Sigma-Aldrich). After washing the membrane, added the secondary antibody, incubated for 50 minutes at room temperature on the shaker. At the end, the membrane was washed five times with TBST for 5 minutes each time, and the ECL was exposed. Gelpro 32 analysis was performed for gel image analysis.

Following stimulations, cells were washed two times with PBS and lysed with either 2% IGEPAL CA‐130 (catalog number I8896; Sigma) in Tris buffer, as previously described,¹ or radioimmunoprecipitation assay buffer (150 mm NaCl, 1% Triton X‐100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mm Tris pH 8.0). Lysates were boiled with Laemmli buffer (under reducing conditions), loaded and separated on 4–12% NuPAGE bis‐tris gels (catalog number NP0322PK2; Novex/Thermo Fisher) and transferred to polyvinylidene difluoride membranes. Membranes were blocked for 1 h with 5% skimmed milk in PBS with 0.1% Tween 20 and stained with primary antibodies overnight at 4°C (1:1000 dilution in 2.5% bovine serum albumin). Primary antibodies were rabbit anti‐Beclin‐1 (catalog number sc‐11 427; Santa Cruz) and mouse anti‐β‐actin (clone AC‐15; Sigma). The membranes were then probed with horseradish peroxidase–conjugated secondary antibodies (diluted 1: 10 000 in 2.5% bovine serum albumin) for 60 min at room temperature. After washing with PBS with 0.1% Tween 20, the blots were developed with enhanced chemiluminescence using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and scanned in a Kodak IS4000R imager (Fisher Scientific).

The Poli^m allele was generated in a C57BL/6 background (Ozgene Pty Ltd.) using standard genetic manipulation techniques. The mutant allele was distinguished from the wild-type allele by PCR amplification and subsequent digestion with TseI restriction enzyme (New England Biolabs). To this end, genomic tail DNA was amplified with forward 5′-ACACACACAATACTACACA-3′ and reverse 5′-AAGCTGCTGGAGTCTTTT-3′ primers to generate a 320-bp fragment. The product was then digested with TseI, and separated on an agarose gel to visualize products. The 320-bp wild-type band was not cut, whereas the mutant band generated 270- and 50-bp fragments. For Western blots, protein extracts from mouse testes were prepared and analyzed with affinity-purified rabbit anti-Pol ι (McDonald et al., 2003 (link)) and mouse anti–β-actin (clone AC-15; Sigma-Aldrich), followed by goat anti–rabbit or goat anti–mouse IgG peroxidase antibodies (Sigma-Aldrich), respectively. Chemiluminescent detection was performed using ECL plus Western blotting substrate (Thermo Fisher Scientific).
Homozygous Poli^m/m and C57BL/6 mice were bred in the NIA mouse facility. 129X1/SvJ mice were purchased from The Jackson Laboratory. Mice of both genders were used at 2–6 mo of age. All animal procedures were reviewed and approved by the Animal Care and Use Committee of the National Institute on Aging.