WBAntibody00002918

Anti-rHc-DHS-28 polyclonal antibody was produced by immunising Institute of Cancer Research (ICR) mice (5 weeks old, female) thrice with 0.5 mg/kg of purified rHc-DHS-28 every 7 days and the titer was determined by ELISA. dhs-28

WBAntibody00002705

Peptide-derived polyclonal antibodies were generated in rabbits against subunit UNC-29.1 (21st Century Biochemicals-Marlborough, MA). Animals were immunized with a mixture of two specific peptides corresponding to parts of the intracellular loop located between the third and the fourth TM domains. For UNC-29.1, the synthetic peptides were 'EKTASPKTLNCAMELTTRDPQL' and 'PIPEPAPPEITQLP'. Purified antibodies were tested for specificity and titrated by ELISA. unc-29

WBAntibody00002706

Peptide-derived polyclonal antibodies were generated against subunits UNC-29.2 in rats (21st Century Biochemicals-Marlborough, MA). Animals were immunized with a mixture of two specific peptides corresponding to parts of the intracellular loop located between the third and the fourth TM domains. For UNC-29.2, the synthetic peptides were 'VSIKVLNCGDDIPKDP' and 'MTKKKRGATVAKLP'. Purified antibodies were tested for specificity and titrated by ELISA. unc-29

WBAntibody00003021

Rabbits were initially immunized with 200 μg full-length His::TEV tagged PALS-5 antigen in Freund's Complete Adjuvant. Rabbits were then subsequently boosted with four separate immunizations of 100 μg antigen in Freund's Incomplete Adjuvant over a 16-week period. Approximately 25 ml of serum was collected and PALS-5 polyclonal antibody was purified with an immuno-affinity chromatography column by cross- linking PALS-5 to cyanogen bromide (CNBr)-activated Sepharose 4B gel. Antibody was eluted from the affinity column in 100 mM glycine buffer pH 2.5, precipitated with polyethylene glycol (PEG), and concentrated in PBS pH 7.4 + 0.02% sodium azide. Antibody concentration was determined by ELISA. pals-5

WBAntibody00002847

The murine monoclonal against phosphorylated S58 of FEZ1 was generated following the standard protocol from Synaptic Systems (Gttingen, Germany). Briefly, three 8- to 10-week-old female BALB/c mice were immunized over a period of 17 days with an FEZ1 phospho-peptide (EIISFKpSMEDL) coupled to KLH. Cells from knee lymph nodes were fused with the mouse myeloma cell line P3 63Ag.653 (ATCC CRL-1580). Cell culture supernatants obtained from individual clones were then screened using an enzyme-linked immunosorbent assay (ELISA) and immunoblot assays to ascertain their speci city towards phospho-58. e nal hybridoma used in this study was cloned two times by limiting dilution. e monoclonal antibody produced from this clone was determined to be of the IgG2a subclass.

WBAntibody00002753

A rabbit phospho-specific polyclonal antibody was generated by Abmart using the phosphopeptide C-QFDR(pS)FILAS encompassing the S269 site in SYP-4. Two peptides were synthesized: An antigen peptide with phosphoserine C-QFDR(pS)FILAS and a control peptide without phosphorylation (C-QFDRSFILAS). Serum harvested from the rabbits immunized with the antigen peptides went through two rounds of affinity purification. First, serum was passed through a column to which the antigenic peptide was coupled in order to isolate phospho-specific SYP-4 antibodies. The eluate from the first column was then passed through a second column to which the control peptide was coupled and flow through was collected. This second step was done to remove any non-phosphorylated SYP-4 antibodies. An ELISA titer of ≥ 1:50,000 against the modified peptide and a modified/unmodified titer ratio of ≥ 8 were used as validation criteria. syp-4