Mini gel system

R1 cells were grown in 6-well plates seeded with CD1 MEFs as feeders, and treated by lentiviruses as described in the Apoptosis and cell cycle assays section. Total cellular proteins were then extracted using RIPA buffer (Thermo Fisher Scientific) with 1× proteinase and phosphatase inhibitors (Thermo Fisher Scientific). Proteins were quantified with a BCA-Quantification kit (Thermo Fisher Scientific), and subjected to 10% SDS-PAGE gel electrophoresis using BioRad mini-gel system and subsequently transferred to PVDF membranes.
The blotted membranes were then blocked with 5% non-fat dry milk in TBS-T and incubated with primary antibodies at 4°C overnight. The antibodies used were as follows: anti-GAPDH (1/2500, Abcam). Antibodies against p53, Mdm2, pMdm2, and pGSK3β (1/1000 for all) were from Cell Signaling. All Akt isoforms, phospho-Akt Ser473, and pan-Akt were detected using Akt isoform antibody sampler kit from Cell Signaling. Membranes were then washed and blotted with HRP conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Chemiluminescence was detected either using Pierce ECL Western-Blot Substrate (Thermo Fisher Scientific) and X-ray film exposure, and quantified using the ImageJ software (Schneider et al., 2012 (link)), or detected and quantified using BioRad Gel Doc XR System (Hercules, CA, USA).

Standard Western blot procedure was using throughout the study. SDS-PAGE was carried out using the mini gel system from Bio-Rad. Proteins in gels were then transferred to PVDF membrane. After blocking with TBST containing 5% non-fat dry milk, the membrane was incubated overnight with primary antibodies diluted with TBST containing 5% BSA using dilutions suggested by the manufacturers. After thorough wash with TBST, the membrane was further incubated with horse radish peroxidase-conjugated secondary antibodies for 1 hour at room temperature followed by thorough washing with TBST buffer. The signals on the membrane were developed with an ECL system (Pierce).

Cells were washed twice with PBS and then lysed in ice-cold T-PER buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Milipore Sigma). SDS-PAGE was carried out using the minigel system from Bio-Rad, and proteins were transferred to PVDF membranes. After blocking with TBST containing 5% nonfat dry milk for at least one hour at room temperature, the membranes were incubated at 4°C overnight with primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for one hour at room temperature. After thorough washing with TBST buffer, signals on the membranes were developed with an enhanced chemiluminescent system (Pierce). Antibodies used in this study include the following: scleraxis (Abcepta #AP21316b, 1 : 1000), tenomodulin (Santa Cruz Technology #sc-49325, 1 : 1000), Mohawk (Abcam #ab179597, 1 : 1000), α-tubulin (Cell Signaling Technology #3873, 1 : 1000), p-SMAD3 (Santa Cruz Technology #sc-517575, 1 : 1000), and p-SMAD1/5 (Cell Signaling Technology #9516T, 1 : 1000).

Samples were homogenized in RIPA buffer and the homogenized samples were centrifuged for 5 min to isolate the total proteins. Next, the proteins were separated by standard SDS-PAGE using the Mini-gel system (Bio-Rad) and transferred to a nitrocellulose membrane by the semi-dry transfer apparatus (Bio-Rad). Membranes were incubated in the blocking buffer at ambient conditions for 1 h followed by incubation with primary antibodies (1:1000) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000) for 1 h at ambient conditions. After extensive washing with a mixture of tris-buffered saline and Tween-20 (TBST), the membrane was incubated with ECL detection reagents and exposed to an X-ray film. The intensity of the bands was quantified with the Image-J software (National Institutes of Health, Bethesda, MD).

Proteins were usually separated by standard SDS-PAGE (12% acrylamide) using the minigel system (Bio-Rad). Proteins were denatured by 5 min boiling in Laemmli-buffer containing 70 mM SDS and 2.4 mM 2-mercaptoethanol. The separated proteins were either stained with Coomassie-brilliant blue or transferred onto PVDF membranes (Hybond, Amersham) using electro-blotting for subsequent protein detection with specific antibodies.
Soluble protein extracts (10 μg) from cells of WT, ΔacnSP, or pVZ322_AcnSP, which were cultivated at 100 μmol photons m^–2 s^–1 of continuous light, were used for Western-blotting. For the detection of aconitase a peptide antibody was generated. The synthetic peptide (Ac)-CELLKNPPEAKEEL-amidated specific for AcnB of Synechocystis 6803 was used to produce antiserum in rabbits (Agrisera AB, Sweden). The FLAG-tagged AcnSP protein version in strain pVZ322_AcnSP was detected with a commercial anti FLAG-tag antibody [Monoclonal ANTI-FLAG M2-Peroxidase (HRP), Sigma-Aldrich, United States].