Goat immunoglobulins

To generate polyclonal antibodies against hPCL3L and hPCL3S, the N-terminal common peptide corresponding to AA 1-15 of human hPCL3 (NH2-MENRALDPGTRDSYG+C-CONH2) was synthesized, coupled to KLH and used to immunize rabbits (Eurogentec, Belgium). Specific antibodies were purified by affinity chromatography using standard protocols. For hPCL3S, we also used commercial antibodies generated against a GST-hPCL3S fusion protein (Proteintech, rabbit 11895-1-AP) or against a C-terminal peptide (Everest biotech, goat EB22188).
Commercial primaries antibodies of the following specificities were also used: EZH2 (Cell Signaling, 5246), SUZ12 (Cell Signaling, D39F6), α-tubulin (Santa Cruz, sc-23948), GAPDH (Santa Cruz, sc-32223), Lamin (Santa Cruz, sc-20681), β-catenin (Santa Cruz, sc-7199), Vimentin (Santa Cruz, sc-6260), AM-Tag: (Active Motif, 61677) and normal rabbit IgG (Cell Signaling, 2729). Western blots were performed as previously described [48 (link)]. The secondary antibodies were horse-radish peroxydase-linked antibodies against rabbit, rat and mouse immunoglobulins (Amersham Biosciences) or goat immunoglobulins (Southern Biotech).

Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes (GE healthcare). Western blot analyses were performed as previously described [8 (link)].
Commercial antibodies of the following specificities were used: FLAG M2 from Sigma; Anti MTA1 (sc-9445 for WB and sc-10813 for IP) from Santa Cruz and Anti MTA3 (ab87275) from Abcam; γH2AX, H2AX (total), pS1681ATM, ATM total, DNA-PKcs total, DNA-PKcs (pS2056) from Abcam, Chk2, pS516Chk2, pT68Chk2 pS20P53 and anti-actin antibodies (sc-1616-R) from Santa Cruz Biotechnology. The secondary antibodies were horseradish peroxidase-linked antibodies against rabbit, rat and mouse immunoglobulins (Amersham Biosciences); goat immunoglobulins (Southern Biotech).
To analyze the SUMOylation of HIC1 proteins by Western blotting analyses, transfected HEK293T cells pelleted by centrifugation were directly lysed in Laemmli loading buffer, boiled for 10 minutes and processed for Western blotting as described above.

Commercial primary antibodies of the following specificities were used: HIC1 (mouse monoclonal antibody against HIC1 C-terminal Amino Acids 611-733: H-6 Santa Cruz, sc-271499, dilution 1/200), Lamin (Santa Cruz, sc-20681), E-cadherin (Santa Cruz, sc-7870), Vimentin (Santa Cruz, sc-6260) and α-SMA (ab5694, Abcam). Western blots were performed as previously described [25 (link)]. The secondary antibodies were horse radish peroxidase-linked antibodies against rabbit and mouse immunoglobulins (Amersham Biosciences) or goat immunoglobulins (Southern Biotech). Immunofluorescence analyses were performed essentially as previously described [52 (link)] on WPMY-1 cultured on coverslips using anti-HIC1 H-6 (dilution 1/50) and Alexa Fluor 488 goat anti-mouse IgG (Life Technologies) as secondary antibodies.