Gateway system

For knockdown of Nestin expression, retrovirus vectors (pSM2) encoding shRNAs were purchased from Open Biosystems (Huntsville, AL, United States) (Yang et al., 2017 (link)). In addition, we used lentivirus provided by Andy Peng Xiang to upregulate Nestin. Myc-tagged Nestin was constructed using Invitrogen’s Gateway System. For overexpression of HIF1-α, plasmid was constructed using Invitrogen’s Gateway System. MG132 and CHX were purchased from Sigma. Non-target control (NTC) was used as the control group.

To generate the Sbm-Gal4 transgenic line we analyzed the genome region of the sbm gene with Evoprinter (https://evoprinter.ninds.nih.gov/), comparing to the D. sechellia, D. yakuba, D. erecta and D. Pseudoobscura sequences and found that there were sections of the introns of the gene with a degree of conservation that suggested that they could contain regulatory sequences. We cloned into the TOPO vector (Invitrogen) an approximately 3.4 kb long fragment of the locus including the introns of the RC and RD transcripts, from the template BACR22I24 (Genebank ID AC022346, obtained from the BAC PAC Resources at the Children’s Hospital Oakland Research Institute) with the primers listed in Table S1. Using the Gateway system (Invitrogen) we subcloned this fragment into the pBPGAL4.2Uw-2 (a gift from Gerald Rubin, Addgene plasmid #26227, (Pfeiffer et al., 2010 (link)) and sent to Genetic Services, Inc. (MA, USA) for injection into their y, w; attP2 line (location 68A4, chromosome 3L.)
To generate the UAS-Sbm transgenic line, we used the Gateway system (Invitrogen) to subclone the CDS from the TOPO vector to the pUASg:attB vector (Bischof et al., 2013 (link)) and sent to Genetic Services, Inc. (MA, USA) for injection into their y, w; attP2 line (location 68A4, chromosome 3L.)

Human EphA2 cDNA was purchased from Open Biosystems (Huntsville, AL, USA). Gene fragments corresponding to nucleotides 1–981 and 1–1578 of the coding region (antigens #1 and 5) were amplified using the polymerase chain reaction, fused to a FLAG-tag-coding sequence at the 3′ end, and subcloned into the pcDNA3.2-DEST mammalian-expression vector using the Gateway System (Invitrogen, Carlsbad, CA, USA; Figure 1). Expression vectors for the EphA2 ligand-binding and cysteine-rich domains (antigen #1) and EphA2 extracellular domain (antigen #5) were transiently transfected under serum-free conditions into FreeStyle 293-F and A431 cells, respectively. These recombinant antigens were secreted into serum-free conditioned medium, collected, and purified by anti-FLAG mAb-conjugated agarose-affinity chromatography as described previously.^{25 (link)} Purity of the recombinant proteins was verified by SDS-PAGE with Coomassie Brilliant Blue staining.
For antibody epitope mapping, antigens #2, 3, and 4, corresponding to EphA2 aa 28–125, 101–250, and 226–328, respectively, with C-terminal FLAG tags, were amplified using the polymerase chain reaction and subcloned into the pET160 vector using the Gateway System (Invitrogen; Figure 1). Recombinant antigens were expressed in E. coli and purified with anti-FLAG mAb-conjugated agarose chromatography as described previously.^{21 (link)}

Adenoviruses were prepared using the ViraPower Adenoviral Expression System (Invitrogen), as previously described^{1,19, 20,21}. PCR-amplified, C-terminal 6xHis and 3xFLAG tagged full-length Ildr2 was subcloned into the pENTR/D-TOPO vector using the pENTR Directional TOPO Cloning Kit (Invitrogen). Inserts of pENTR-Ildr2-6xHis-3xFLAG vector were transferred into the pAd/CMV/V5-DEST vector by the Gateway system (Invitrogen).
For the shRNAs of Ildr2 and LacZ were cloned into BLOCK-iT U6 entry vector (Invitrogen). The sequence of the shRNA for Ildr2 shRNA1 was: 5′- cacc GAAGAAGGTGGCCATGCTC acgtgtgctgtccgt GAGCATGGCCACCTTCTTC -3′ and Ildr2 shRNA2 was: cacc GCTGATTTCAAATCTTAGT gcgcttcctgtcacgc ACTAAGATTTGAAATCAGC. The inserts of pENTR shRNAs vectors were transferred into the adenovirus vector pAd/PL-DEST using the Gateway system (Invitrogen). The recombinant adenoviruses were purified by the Adenovirus Purification Miniprep Kit (Cell Biolabs).

A previously described construct with the Col-0 genomic FLM fragment pFLM::gFLM template (pDP34) (Posé et al., 2013 (link)) was recombined into a pDONR201 destination vector using the Gateway system (Life Technologies, Carlsbad, CA). This vector was used as a template (FLM^Col-0) to generate mutations using either a single phosphorylated primer or a combination of forward and reverse primers (Sawano and Miyawaki, 2000 (link); Hansson et al., 2008 (link)). In case of variants with multiple modifications, individual mutations were introduced one at a time. The mutated inserts were recombined to the pFAST-R07 expression vector using the Gateway system (Life Technologies, Carlsbad, CA) (Shimada et al., 2010 (link)). All expression constructs were verified by sequencing and transformed into Agrobacterium tumefaciens strain GV3101. Nd-1 plants were transformed using the floral-dip method (Clough and Bent, 1998 (link)) and transgenic plants were identified based on seed fluorescence (Shimada et al., 2010 (link)). Segregation of T2 lines was examined and lines with single insertion events were selected for further analysis based on segregation ratios (Shimada et al., 2010 (link)). A list of primers and expression constructs is listed in Supplementary file 7.