Optimumantigen design tool

Plasmids expressing miR162a were transfected into S2 cells using Effectene (Qiagen) according to the manufacturer’s instructions. The cells were lysed in RIPA buffer (0.5% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5)). The lysates were resolved via 6% SDS-PAGE (for the dmTOR protein) or 10% SDS-PAGE (for internal control GAPDH protein), transferred to a PVDF membrane (Millipore, Bedford, MA, USA) and probed with anti-dmTOR or anti-GAPDH antibodies (Santa Cruz Biotechnology, CA, USA). Anti-dmTOR antibodies were polyclonal antibodies that were custom-made by GenScript USA Inc. (Nanjing, China). The epitope was predicted using the GenScript OptimumAntigen design tool, and the peptide antigen was then synthesized. After the coupling reaction and mixing with complete adjuvant, the coupled antigen was used once for a subcutaneous injection. The host strain was a New Zealand rabbit. Then, the coupled antigen was mixed with incomplete adjuvant and injected into the rabbit. Subsequently, serum was taken from the immunized rabbit, and the antibody was purified.

For immunoblot analysis, total membrane proteins (25 μg) resolved by SDS-PAGE under nonreducing (in the absence of β-mercaptoethanol) conditions (37 (link)) as described above were electrotransferred onto an Immun-Blot polyvinylidene difluoride (PVDF) membrane (Bio-Rad) and probed for LptD or LptE using anti-LptD (1:20,000) and anti-LptE (1:30,000) polyclonal rabbit antibodies (GenScript) and goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma-Aldrich). Anti-LptD and anti-LptE antibodies were generated at GenScript using keyhole limpet hemocyanin (KLH)-conjugated peptides as the antigens (LptD epitope, QLHQKEAPGQPEPVC, and LptE epitope, ELLDKETTRKDVPSC). The epitopes were selected using the OptimumAntigen design tool (GenScript). The transferred membrane was developed with Clarity Western ECL substrate (Bio-Rad) and visualized in a FluorChem Q imager (ProteinSimple).

The antibody of AtPAP9 was produced by PolyExpress™ Antibody Services (GenScript, Nanjing, China). The AtPAP9-specific peptide (a.a. 450–468, YTTSRKIRDAAIREKMIEH) designed using the OptimumAntigen design tool (GenScript, Nanjing, China) was used to immunize rabbits (Figure S2). The antibodies (anti-AtPAP9) were affinity-purified by the peptides. Anti-PAP2 antibodies were generated, as previously described [8 (link)]. Horseradish peroxidase (HRP)-labeled secondary antibodies were used, and protein bands were visualized using the WesternBright^TM Quantum Western blotting detecting kit (Advansta, San Jose, LA, USA).

Recombinant M. synoviae Obg was expressed in, and purified from, E. coli as Obg-MBP and used to inoculate chickens for polyclonal anti-Obg-MBP chicken serum (S2 File). Using GenScript OptimumAntigen design tool, several highly hydrophilic/antigenic epitopes were predicted in M. synoviae Obg. Based on criteria for surface probability, homology to mouse or rabbit proteins, flexibility, secondary structures, signal peptide and solubility, four peptides (Obg_1 TSHLQIKIEDDFET, Obg_2 GKGGRGNNKFKTSK, Obg_3 NTAPRIAENGMPGE and Obg_7 KMNRNHFKVTGKKI) were selected and synthesized (GenScript, Piscataway, New Jersey, USA). New Zealand rabbits were immunized with each of these peptides and affinity-purified polyclonal antibodies were produced (GenScript) and used in immunoblotting as described below.

Custom-made polyclonal antibodies were used. The epitopes were predicted using the GenScript OptimumAntigen design tool, and the peptide antigen sequences for AccCYP314A1, AccCYP4AZ1, and AccCYP6AS5 were PFGAGRRICPGK, EAHRNNKIDDEGIRE, and PNPDSFDPERFDQDAMAS, respectively. The peptide antigens were then synthesized (Sangon, China). After the coupling reaction and mixing with complete adjuvant, the coupled antigen was used for injection. Next, the coupled antigen was mixed with incomplete adjuvant (Sigma, United States) and injected into white mice (Taibang, China), which were 6 weeks old and specific pathogen-free, four times at 4-week intervals. Subsequently, blood was collected by eyeball puncture, incubated at 37°C for 1 h, and centrifuged at 3,000 × g for 10 min. Finally, the anti-serum was prepared. The collected antibody was hybridized to a blot containing the overexpressed proteins to detect the specificity of the anti-serum.

The epitope of anti-Huntingtin Ab of Sp-Htt was NH2-C+²⁷⁴TSPATSPQEGEGS²⁸⁷-COOH in the amino acid sequence (SPU_012067.1) registered in EchinoBase (http://legacy.echinobase.org/Echinobase/AboutEB) (Access on: 3 March 2021), and was chosen based on GenScript’s Optimum Antigen design tool (https://www.genscript.com/antigen-design.html) (Access on: 3 March 2021) analysis. The sequence also was reported in the earlier report on Sp-Htt [14 (link)]. The homologous amino acid sequence is also present in Hp-Htt [Hp-Hunt (middle), HPU_11725], as well as with one peptide difference at ²⁸³G in S. purpuratus and ¹⁴¹A in H. pulcherrimus (Figure 7).
The antigen, NH₂-CTSPATSPQEGEGS-COOH, was synthesized and utilized for raising antibody in rabbits by Eurofins Genomics K.K (Ota-ku, Tokyo, Japan 143-0003). Before inoculation, blood serum was collected from the rabbits and used as a pre-immune serum.