Gel logic 2200

Purified GST-CpnA samples were thawed on ice and spun at 150,000× g (50,000 RPM) for 1 h at 4 °C. The supernatant was removed and kept on ice. An F-actin stock was prepared as described in the published protocol (Cytoskeleton, Inc.). Three samples were prepared for the F-actin binding assay: (1) An F-actin control sample made by mixing 40 µL F-actin stock (1 mg/mL) and 10 µL of glutathione elution buffer; (2) a no actin protein control made by mixing 10 μL GST-CpnA protein (20 μg/mL) and 40 μL F-actin buffer; and (3) an experimental sample made by mixing 40 μL F-actin stock (1 mg/mL) and 10 μL GST-CpnA protein (24 μg/mL). In addition, the same three samples were prepared again, but 2 mM EGTA was added to the samples. All samples were incubated at RT for 30 min then spun at 150,000× g in an Airfuge for 1.5 h at 24 °C. The supernatants were removed, and 10 μL 6× sample buffer was added into each supernatant. The pellets were resuspended with 30 µL 2× sample buffer. All the samples were analyzed by SDS-PAGE and stained with Coomassie Blue. A western Blot was performed for the supernatants and pellets from the samples that included GST-CpnA using an HRP conjugated anti-GST antibody (1:5000). An ECL chemiluminescence kit (Amersham Biosciences) and the Kodak Gel Logic 2200 were used to image the blot.

Untransfected HeLa, 5637, 1321, A172, NCCIT and NT2 cell cultures were lysed with 1 ml of lysis buffer [1% Triton X-100, 5 mM EDTA, 50 mM TrisHC1, pH=7.4, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS)], 1% protease inhibitor cocktail and phosphatase inhibitor (Sigma, USA). Protein concentrations were measured by the Bradford Protein Assay reagent. Briefly, 20 µg of cell lysates were isolated on 12.5% SDS-PAGE and transferred onto a Polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Blocking was carried out with 5% skim milk in tris-buffered saline (Sigma-Aldrich, Germany), containing 0.05% Tween20 (TBS-T). The membrane was then incubated for 1 hour with primary mouse anti-Oct-3/4 sc-5279 antibody (Santa Cruz, USA) diluted in 3% blocking buffer, and washed with PBS for 30 minutes at room temperature. A secondary HRPconjugated sheep-anti-mouse antibody (Avicenna Research Institute, Iran) was added next and incubated at RT for 1 hour. After washing, signals were detected using the Amersham ECL Prime Western Blotting Detection kit (GE Healthcare Life Sciences) and quantified by Gel Logic 2200 (Kodak, Japan).

Purified RNA samples underwent reverse transcription to synthesize cDNA using the Qiagen QuantiTect Reverse Transcription kit following manufacturer's instructions. Control reactions without reverse transcriptase were also performed for each sample. Reverse transcriptase PCRs (RT‐PCRs) were then performed using 2 μl of each reverse transcription (RT) reaction mix using gene‐specific primers described in Table A2. A control without reverse transcriptase enzyme in the RT reaction mix was used for each PCR. Fragments were resolved by electrophoresis on agarose gels. At least three replicates from each biological replicate were created. Pictures of gels were taken under UV light with a Kodak Gel Logic 2200 imaging system at an exposure time of 0.1 s. Images were then analyzed using ImageJ software. Numerical values for each gene could then be normalized to our control gene glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) for each band.