Anti β tubulin ab6046

Pulverized ankle joints or synovial fibroblasts were resuspended in lysis buffer (0.1 M Tris-HCl, 0.2 M EGTA, 0.2 M EDTA, 100 mM sodium orthovanadate, 50 mM sodium fluoride, 100 mM sodium acid pyrophosphate, 250 mM sucrose, pH 7.5) and total protein (60 μg), subjected to SDS/PAGE, blotted, and probed overnight with 1:1000 anti-PRL receptor (sc-300; Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1:250 anti-phospho-STAT3 (Tyrosine 705) (9131; Cell Signaling, Beverly, MA, USA), 1:250 anti STAT3 (sc-483; Santa Cruz Biotechnology), or 1:1000 anti-β tubulin (ab6046; Abcam, Cambridge, MA, USA) primary antibodies. Blots were washed in Tris-buffered saline/Tween-20 and detection was performed using goat anti-rabbit conjugated to alkaline phosphatase (1:5000) or to horseradish peroxidase (1:10,000) as secondary antibodies (both from Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). Protein densities were quantified with Quantity One software (Bio-Rad, Richmond, CA, USA).

Protein lysates were prepared as above, denatured using 10 × NuPage Sample Reducing Agent (ThermoFisher, Waltham, MA, USA), and run on NuPAGE Novex 4–12% Bis–Tris precast gels (Invitrogen) in MES (2-(N-morpholino) ethanesulfonic acid) buffer (ThermoFisher). Proteins were transferred using the iBlot® Dry Blotting System (Invitrogen) and signal detected using the Li-Cor® Odyssey Infrared Imager (Li-Cor, Lincoln, NE, USA). The following antibodies were obtained from Cell Signaling Technologies (Danvers, MA, USA): pERK p44/42 MAPK T202/Y204 (9106); t-ERK p44/42 MAPK (9107), p-ERBB3 Tyr1289 (2842S), ERBB3 (4754S), p-ERBB2 Tyr 1248 (2247), ERBB2 (2242), pEGFR Tyr1068 (D7A5) and EGFR (2232). Anti-β-tubulin (ab6046) was obtained from Abcam (Cambridge, UK) and anti-β-actin (A5316) from Sigma.

Resveratrol (RES; R5010), 3-methyladenine (3-MA; M9281), EX-527 (E7034), wortmannin (WM; W1628), and STO-609 were purchased from Sigma-Aldrich (Saint Louis, MO, USA), and Compound C (171260) was purchased from Calbiochem (San Diego, CA, USA). SYBR Green reagents were purchased from Applied Biosystems (Waltham, MA, USA). Dimethyl sulfoxide (DMSO; Sigma-Aldrich, Saint Louis, MO, USA) was used at 0.1% (v/v) as a solvent control.
Specific antibodies against AMPKα (2532), FoxO3a (75D8), FoxO1 (C29H4), phospho-Fox01 (9464), phospho-AMPK (2535), phospho-CAMKK2 (12818), phospho-LKB1 (3482), and acetylated-Lysine (9441) were purchased from Cell Signaling (Danvers, MA, USA). Phospho-Fox03a (AF2343) was purchased from Affinity Biosciences. Anti-LC3 (L8918) was obtained from Sigma-Aldrich. Anti-β-tubulin (Ab6046) and anti-SIRT1 (Ab28170) were purchased from Abcam (Burlingame, CA, USA). Anti-TP3 (sc-52255) and anti-LAMP1 (sc-17768) were purchased from Santa Cruz (Dallas, TX, USA). All other reagents were obtained from Sigma-Aldrich, unless otherwise indicated.