Goat anti actin antibody

For the immunoblot analysis, 20 nM CKD-5 was treated 24 h before, followed by treatment with 2 μM sorafenib. Cells were lysed on ice for 20 min using lysis buffer (50 mM Tris-HCl, pH 7.4; 1% Nonidet P-40, 0.25% sodium deoxycholate; 150 mM NaCl; 1 mmol/L EDTA; 1 mM phenylmethylsulfonyl-fluoride; 1 mM Na3VO4; 1 mM NaF; and 1 mg/mL each of aprotinin, pepstatin, and leupeptin) and centrifuged at 14,000×g for 10 min at 4 °C. 50 μg and 30 μg of protein from SNU761 and SNU3058 cells, respectively, were loaded on a 12.5% SDS-PAGE gel. Samples were transferred to nitrocellulose membranes, blotted with appropriate primary antibodies at a dilution of 1:1000, and treated with peroxidase-conjugated secondary antibodies (Biosource International, Camarillo, CA, USA). Bound antibodies were visualized using chemiluminescent substrate (ECL; Amersham, Arlington Heights, IL, USA) and exposed to Kodak X-OMAT film (Kodak, New Haven, CT, USA) [25 (link)]. Primary antibodies included rabbit anti-caspase 9 and 7 (cleaved) (Cell Signaling Technology, Danvers, MA, USA), anti-heat shock protein 90 (HSP90), anti-P21 (both from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-GPX4, and anti-acetylated H3 (both from Abcam, Cambridge, UK). A goat anti-actin antibody was also used (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Cells were lysed for 20 min on ice with lysis buffer and centrifuged at 14,000× g for 10 min at 4 °C. Samples were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, blotted with appropriate primary antibodies at a dilution of 1:1000, and treated with peroxidase-conjugated secondary antibodies (Biosource International, Camarillo, CA, USA). Bound antibodies were visualized using chemiluminescent substrate (ECL; Amersham, Arlington Heights, IL, USA) and exposed to Kodak X-OMAT film (Kodak, New Haven, CT, USA). Rabbit anti-regenerating gene protein-3A (REG3A) was obtained from Abcam (ab95316). Primary antibodies including rabbit anti-phospho-p42/44 mitogen-activated protein kinase (MAPK), anti-phospho-Akt, rabbit anti-c-myc, rabbit anti-caspase 8, anti-caspase 9, and anti-caspase 7 (cleaved) were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat anti-actin antibody was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Densitometric analyses were performed using Image J software (National Institutes of Health, Bethesda, MD, USA).

A total of 30 guts were dissected from 5-, 30- and 60-day-old flies, or Myo1A^ts>+ and Myo1A^ts>PEBP1 RNAi flies after induction of 5 days at 29° C. Total protein from these guts were extracted using a lysis buffer (Intron Biotechnology, Korea) with a protease inhibitor (Roche, Basel, Switzerland). Protein quantification was done with Bradford reagent (Bio-Rad) before equal amount of protein was loaded. The whole gut extracts were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gels containing 15% acrylamide and the protein bands were transferred onto polyvinylidene fluoride membranes (GE Healthcare Life Sciences, England). The blotted membranes were blocked with 1×TBST containing 5% skim milk, followed by their overnight incubation with the rabbit PEBP1 antibody (1:2000) and goat anti-actin antibody (1:5000; Santa Cruz Biotechnology, Dallas, TX, USA) at 4° C. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000) for 2 h at room temperature. The protein bands were detected with enhanced chemiluminescence (ECL) western blotting detection reagent (ThermoFisher Scientific, MA, USA).

Cells were lysed for 20 min on ice with lysis buffer and centrifuged at 14,000 g for 10 min at 4°C. Samples were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, blotted with appropriate primary antibodies at a dilution of 1:1000, and treated with peroxidase-conjugated secondary antibodies (Biosource International, Camarillo, CA). We also performed electrophoresis of protein extracts derived from keloid or normal dermal tissue using a Tris-glycine buffer system, and subsequent blottings were performed. Bound antibodies were visualized using chemiluminescent substrate (ECL; Amersham, Arlington Heights, IL) and exposed to Kodak X-OMAT film (Kodak, New Haven, CT). Primary antibody for rabbit anti-PARP1 was purchased from Cell Signaling Technology (Danvers, MA). Goat anti-actin antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Densitometric analyses were performed with ImageJ software (National Institutes of Health, Bethesda, MD).