Acrylamide gel

Protein concentration of each SF solution was measured using Bradford assay (Bio-Rad laboratories, Hercules, CA, USA) and the solutions were mixed with 2X Laemmle buffer (Bio-Rad) and 2.5% β-mercaptoethanol. Denatured SF samples at 95 °C for 2 min were loaded into the lanes of acrylamide gel (Bio-Rad) and electrophoresis was performed. Next, the gel was stained using a silver staining kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s guide. Using the analyze line graph tool of the image processing software (ImageJ), the gray value profile was created along each lane of the gel image. The representative profile of three replicates was selected and shown herein.

Cells were lysed with CelLytic M (Sigma-Aldrich) or RIPA buffer plus protease and phosphatase inhibitors, as described in immunoprecipitation section, and cellular debris was removed by 15 min centrifugation at 10,000 g. Protein extracts, obtained by cell lysis or immunoprecipitation, were denatured with Laemmli Sample Buffer 4X (LSB, NuPAGE-Novex-invitrogen Life technologies) plus 12% β-mercaptoethanol and with boiling of samples for 10 min at 95 °C. Denatured samples were then subjected to SDS-PAGE electrophoresis on acrylamide gel (BIO-RAD) and subsequent electro blotted to a Nitrocellulose (Amersham biosciences) or Polyvinylidene difluoride (PVDF, Millipore) membrane. Then, the membrane was blocked with 5% nonfat dry milk or BSA (Sigma-Aldrich), and was incubated with primary antibodies overnight, with rotation at 4 °C. Detection was possible thanks to horseradish peroxidase-conjugated secondary antibodies. Chemiluminescent reaction was induced with ECL plus (Millipore) and images were acquired with a digital camera (Fluor Chem SP, Alpha Innotec; Amersham Imager 600, Amersham). Background adjustment and cropping of images were done with ImageJ and Photoshop softwares.

The coding sequences of Dam::CrebB and CrebB alone were cloned into the pBluescript vector. Both plasmids were linearized and used as template for protein production with the TNT(R) T7 Quick Coupled T/T System kit (Promega). The translated proteins were mixed with IRDye 700 CREB consensus oligonucleotide (LI-COR) with or without 10-fold excess of consensus or mutated competitor oligonucleotides and incubated for 30 minutes. The reactions were run on a 5% acrylamide gel (biorad) and the oligonucleotides visualized with an Odyssey FM imaging system (LI-COR).

Whole-worm protein extracts were produced by picking 80 young hermaphrodites (24 h post L4) into a 1.5 ml Eppendorff tube containing 40 μl of 1× TE (10 mM Tris-HCl pH 8, 1 mM EDTA) supplied with complete protease inhibitor (Roche). The samples were subjected to three cycles of freezing/thawing using liquid nitrogen before Laemmli buffer was added to a 1× final concentration, and the samples were boiled for 10 min. Equal volumes of protein extracts were run on a 10% acrylamide gel (Bio-Rad) and transferred onto a nitrocellulose membrane for 1 h at 4 °C and then blocked for 1 h in 5% milk TBST (1% TBS and 0.1% Tween) at room temperature. Primary antibodies, mouse anti-His antibody (GenScript 0.2 μg/ml) and goat anti-Actin (Santa Cruz 1:3000), were diluted in 1× TBST and incubated overnight at 4 °C. The following secondary antibodies conjugated to horseradish peroxidase were used: goat anti-mouse (Jackson Immunoresearch) (1:5000) and donkey anti-goat (Sigma) (1:8000). Secondary antibodies were diluted in 1× TBST containing 5% milk and incubated for 1 h at room temperature.