Trans blot semi dry transfer system

Equal amounts of skeletal muscle protein extract (45 µg) were separated on Mini-Protean TGX precast protein gels (Bio-Rad, CA) and transferred to a PVDF membrane using a Trans-Blot semi-dry transfer system (Bio-Rad, CA). The membranes were blocked using 5% BSA in TBST for phospho-antibodies or 5% non-fat milk for other antibodies for 1 hr at RT. The following primary antibodies were used overnight: pAMPK Thr172 (Cell Signaling Technology #2535, Beverly, MA), AMPK (Cell Signaling Technology #2532), GAPDH (Cell Signaling Technology #2118), PGC1a (H300, Santa Cruz Biotechnology #sc-13067), and Beta-Actin (Sigma-Aldrich, #A1978), washed in TBST, and incubated with the respective HRP-conjugated secondary antibodies (Vector Labs, #PI:1000 and #PI:2000, Burlingame, CA) for 1 hr at RT. Proteins were detected (Supersignal West Pico or Femto Chemiluminescent substrate solutions [Thermo Fisher Scientific]) and digital images acquired (GE ImageQuant Gel Doc Imaging system). Densitometric analysis was carried out using Fiji software.

The cells were washed twice with Ca/Mg-free PBS and then treated with a microtubule lysing buffer consisting of 100 mM PIPES, 5 mM MgCl₂, 1 mM EGTA, 30% glycerol, 0.1% IGEPAL, 0.1% Tween-20, 0.1% Triton X-100, 0.1% beta-mercaptoethanol, 1 mM ATP, 0.1 mM GTP and a complete protease inhibitor cocktail (Roche); the recipe is according to Cytoskeleton, Inc. (USA). The lysate was homogenized by Retsch Mixer Mill at 25 Hz for 2 min, and incubated for 30 min at 35 °C. The obtained cell lysates were clarified by centrifugation at 21000 x g for 40 min at 35 °C. The protein concentration in lysates was determined using the Pierce BCA Protein Kit. Proteins were separated by 12% SDS-PAGE and transferred onto the PVDF membrane by Trans-Blot Semi-Dry Transfer system (Bio-Rad, Inc., USA).
To determine the presence of beta-tubulin isotypes Abcam mono- and polyclonal antibodies (anti-beta I Tubulin (ab11312), anti-Tubb2A (ab170931) and anti-beta III Tubulin (ab52901) were used. After the chemiluminescence reaction, the PVDF membranes were stained with Coomassie brilliant blue R250 to measure the total protein amount. The tubulin signal intensity was normalized against total protein intensities obtained from Coomassie staining. Quantification was performed by ImageJ software.

Cells were washed twice with Ca/Mg-free PBS and then treated with a Tris-Triton X lysis buffer consisting of: 10 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 0,1% SDS) supplemented with protease inhibitor cocktail (Roche). Lysates were homogenized by a Retsch Mixer Mill at 25 Hz for 2 min, incubated for 20 min on ice, and clarified by centrifugation at 21,000 g for 30 min at 4°C. The concentration of the isolated proteins is determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, Rockford, U.S.A). Protein samples (35 µg) were separated by a 12% Tris-Glycine SDS-PAGE and electrophoretically transferred onto Immobilon® -P PVDF membrane pore size 45 µm (Merck Millipore, Tullagreen, Ireland) by Trans- Blot Semi-Dry Transfer system (Bio-Rad). Membranes were then incubated with the primary antibodies against AK2 (sc374095) or AK6 (10544-1-AP, Proteintech) and α-tubulin (ab7291) and the HRP conjugated secondary antibodies Goat Anti-Mouse IG (ab97040) and Goat Anti-Mouse IG (ab6721). Mouse kidney homogenate was used as a positive control for AK2. MCF7 serves as a positive control for AK6 recommended by the antibody manufacturer. Blots were developed using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, U.S.A.) and imaged with Biospectrum Multispectral imaging system (Biosoectrum 510, UVP, Cambridge, UK).

Extraction of detergent-soluble and detergent-insoluble material was performed as previously described [18 ], and cells were harvested in cold lysis buffer (20 mM Trizma base, 150 mM NaCl, 1 mM EDTA, 0.25% NP-40, 0.25% Triton X-100, pH 7.4) with protease and phosphatase inhibitors. After 20-min incubation on ice, lysate was spun for 20 min at 4 °C, 14,000 g. Supernatant was stored as detergent soluble fraction, while the pellet was resuspended in lysis buffer with added 5% (wt/vol) SDS and sonicated by cup-horn at 60% amplitude, 3 s on/off cycles for 15 s.
For immunoblotting, even protein amounts were loaded and separated on mini-PROTEAN TGX precast 4–15% Bis–Tris gels (#4,568,085, Bio-Rad, Copenhagen, Denmark). Using a Bio-Rad Trans-Blot semidry transfer system proteins were transferred to a nitrocellulose membrane (#1,704,159, Bio-Rad) and blocked for 1 h (PBS, 0.1% Tween 20, 3% BSA) at room temperature. Primary antibody (α-syn-211, sc-12767, Santa-Cruz Biotechnology, Heidelberg, Germany) staining was performed, 1:1000, in blocking buffer at 4 °C overnight on a shaking table. After three washes with (PBS, 0.1% Tween 20) incubation with secondary species specific HRP-conjugated antibody was performed in blocking buffer for 1 h prior to development on a Bio-Rad ChemiDocMP imaging system.

Isolated cells with or without stimulation were washed with phosphate-buffered saline (PBS) and lysed with NP-40 buffer (NaCl 150 mM, Tris 50 mM, Nonidet P-40 1%, sodium pyrophosphate 4 mM) or RIPA buffer (NaCl 150 mM, Tris 50 mM, Nonidet P-40 1%, sodium deoxycholate 0.5%, SDS 0.1%) supplemented with a Protease Inhibitors cocktail (Sigma) plus 1mM sodium vanadate, 1mM sodium fluoride, 1 mM PMSF. Lysed samples were spun down at 10,000 g to remove debris and boiled at 100°C for 10 min, with Tris-Glycine buffer (SDS 2%, Bromophenol Blue 0.01%) containing 0.1 M DTT. Aliquots were separated via SDS-PAGE. transferred onto PVDF membrane using the Trans-Blot semi-dry transfer system (Bio-Rad). Samples probed for phosphorylation were specially blocked with 5% phosphoBLOCKER (Cell Biolabs) before probing with the following antibodies: anti-SHP-1 (Invitrogen), anti-AKT, or anti-S473-pAKT (Cell Signaling). Blots were re-probed with anti-β-actin HRP (Sigma) to control for loading.