Enhanced chemi luminescence (ecl)

Fractions isolated after raft isolation or whole cell lysates were resolved by SDS-PAGE then transferred to PVDF membranes. The membranes were blocked for 30 min in 5% dry milk solution in PBST (PBS with 0.1% Tween20) and incubated with the indicated antibodies. Membranes were developed using ECL or ECL+ according to manufacturer’s protocol (GE Healthcare, Little Chalfont, UK). Densitometry analysis was performed using AlphaEaseFC software (Genetic Technologies, Inc. Miami, FL).

To quantify eIF4E and 4E-BP in the sea urchin unfertilized eggs, total crude protein extracts were prepared by direct dissolution of 20 μl egg pellet (corresponding to 12,000 eggs) with 150 μl of SDS-Fix buffer as described (Belle et al., 2011 (link)). A range of different extract volumes was submitted to SDS-PAGE and Western-blotted together with known amounts of purified SgIF4E or recombinant Sp4E-BP as prepared above. Proteins in the different samples were immuno-revealed using mouse monoclonal antibody directed against eIF4E from rabbit (BD transduction laboratories, Lexington, KY) or rabbit polyclonal antibodies directed against human 4E-BP2 (Rousseau et al., 1996 (link)), a generous gift from Nahum Sonenberg (McGill University, Montreal, Quebec, Canada). The antigen-antibody complex was measured by chemiluminescence using horseradish peroxydase-coupled secondary antibodies according to the manufacturer's instructions (ECL or ECL+, GE Healthcare Life Sciences). Quantification of the bands was done using the ImageJ 1.43j program. Measurement has been performed on extracts from eggs of 9 different females for eIF4E and 4 females for 4E-BP. Protein concentrations were calculated considering an egg volume of 0.3 nl (diameter 80 μM).

To detect FANCD2, whole cell extracts prepared as described previously [47 (link), 48 (link)] were resolved in pre-cast Tris-acetate gels (Life Technologies), transferred onto nitrocellulose membranes and probed with the α-FANCD2 antibody ab2187 (Abcam). Mouse α-CHK1 antibody was from Santa Cruz (Cat. No. sc-8408). Phosphorylation of CHK1 and CHK2 was analyzed with a Phospho-Chk1/2 Antibody Sampler Kit (Cell Signaling, Cat. No. 9931). Other antibodies were: α-PCNA Cat. No. sc-56 (Santa Cruz), α-RPA32 Cat. No. A300-244A (Bethyl Laboratories), α-γH2AX Cat. No. 05-636 (Millipore), α-Nucleolin Cat. No. 396400 (Life technologies). Biotin-conjugated EdU was visualized in dot blots with HRP-conjugated α-biotin antibody, Cat. No. 7075 (Cell Signaling). All proteins were visualized by ECL (Amersham or Thermo Scientific) and quantified using Storm Phosphoimager and ImageQuant software (Molecular Dynamics) or FluorChem Imager (Alpha Innotech), using manufacturer-supplied software. For presentation, images were saved in TIFF format, adjusted for brightness/contrast and cropped using Adobe Photoshop, and assembled into figures in CorelDraw. Brightness/contrast adjustments were made to entire images. In some cases an image of one and the same blot was cut and spliced to delete extraneous lanes or to change the order of lanes.

Lysates from luciferase assay and ChIP were diluted in 2× loading buffer (Biorad, Hercules, CA, USA) with β-mercaptoethanol, denatured for 5 min at 95 °C and separated on a 10% SDS-page gel. After transferring to Hybond ECL nitrocellulose membrane (GE Healthcare, Little Chalfont, UK), the membrane was blocked in PBS with 0.05% Tween-20 (PBST) containing 3% non-fat dried milk for 1 h at RT, after which anti-p63 (Santa Cruz Biotechnology) was incubated for 2 hours at RT in PBST containing 5% BSA. After incubation with peroxidase labeled secondary antibody rabbit anti-mouse (Dako, Glostrup, Denmark) bands were visualized with ECL+ (GE Healthcare). Positive control lysates for endogenous p63 were obtained by pBABE-LIC-ΔNp63α transfected 293T cells. Briefly, ΔNp63α was PCR amplified from pcDNA3.1-ΔNp63α and cloned into pBABE-LIC vector (obtained from Dr. Priyamvada Rai, University of Miami, USA) as described [44 (link)].

Western blot analyses were performed under denaturing and reducing conditions (unless indicated otherwise) using standard procedures. Protein samples were separated by using a 10% SDS gel (Biorad Laboratories GmbH, #4561031) and running buffer (20 mM Tris, 0.2 M glycine, 3.5 mM SDS). Twenty microgram of whole cell lysate were used and supernatant normalized to according cell pellet. Page Ruler prestained protein ladder (Fisher Scientific GmbH, #26616) was used for determining protein molecular weight. Proteins were transferred onto a PVDF membrane via wet blotting (1 h, 100 V, 4°C). Membranes were probed with C1r or C1s specific antibodies (Abcam plc, Cambridge, UK; C1r: N-terminal #ab71652 or C-terminal #ab185212; C1s: #ab155270) diluted 1:2,000 in PBS-0.05% Tween 20 (PBS-T) containing 5% milk (w/v). HRP-conjugated secondary antibody (Dako, Glostrup, Denmark, #P0448) was used 1:10,000 in 5% milk/PBS-T. Protein bands were detected with ECL (GE Healthcare, Vienna, Austria, #RPN3243) and developed on X-ray films (Amersham Hyperfilm ECL, Distributor: VWR, Vienna, Austria). For overexpressed C1r variants, empty vector transfected HEK293T cells were used as negative control.