Pcdna3.1 myc his b vector

The wild-type PLEK2-CDS was PCR-amplified from HEK-293T cDNA using the following primers: forward 5′-TCGGAGCTGCTTCCTGGG-3′ and reverse 5′-CATGTTAGCTTTTTGATAGCTTCA-3′. Then, it was cloned into the pcDNA3.1/myc-His B vector (Invitrogen, Grand Island, NY, USA). The shRNAs targeting human PLEK2 (5′-AAGTGGCACGGTGGTGAAACA-3′) were cloned into pSilencer 4.1-CMV puro vectors (Ambion, Foster City, CA, USA). Retroviral production and infection were performed as the standard procedure. Stable cell lines expressing PLEK2 or shPLEK2 were selected for 10 days with 400 mg/mL G418 or 0.5 mg/mL puromycin, respectively.

The lentivirus vector-encoding human KDM6A (puro-KDM6A) was purchased from GeneChem Inc. (Shanghai, China). The KDM6A catalytic mutations (H1146A, E1148A) were introduced using a QuickMutation™ Site-Directed Mutagenesis Kit (Beyotime, China). The full-length FOXA1, ARHGDIB, upstream transcription factor 1 (USF1), upstream transcription factor 2 (USF2) and transcription factor binding to IGHM enhancer 3 (TFE3) complementary DNAs were amplified from T24 cells and cloned into the pcDNA3.1/Myc-His B vector (Invitrogen, V85520). The KpnI and XbaI fragment from puro-KDM6A was ligated into the pcDNA3.1/Myc-His B vector to generate pc3.1-KDM6A. The primers used for the construction of overexpression vectors are shown in Table S2.
The sequence of KDM6A shRNA (5′-AAGGAAATTCATTTACGACTT-3′) was synthesized by Sangon Biotech (Shanghai, China) and cloned into the lentiviral vector pLKO.1-Puro (Addgene, 8453). The enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) knockdown lentivirus vectors were purchased from GeneChem Inc. (Shanghai, China). Different progressive deletions of the human KDM6A promoter fragments were amplified from T24 cells and cloned into the pGL3-Basic vector (Promega, E1751). The control plasmid pRL-TK was obtained from Promega (E2241).

CNGB3 complementary DNA39 (link) was kindly provided by Dr Hisao Ueyama, Department of Ophthalomology, Shiga University of Medical Science, Japan. cDNA encoding full-length CNGB3 was excised by HindIII and BsrGI and ligated into the HindIII and Asp718I sites of pcDNA3.1/myc-His-B vector (Invitrogen). HeLa cells were transfected with this vector, and expression of CNGB3–MYC/His fusion protein was determined by immunohistochemistry with an anti-MYC antibody and the anti-CNGB3 antibody (clone 3B2) described above. To test binding of z13 peptide to CNGB3–MYC, FITC-z13 was added (5 μg ml⁻¹) to the medium of HeLa cells transfected with the above-described vector and incubated at 37 °C for 15 min. Cells were washed with PBS, fixed with 4% PFA in PBS and observed under a fluorescence microscope.

Human wild-type E-cadherin and various deletion mutants were constructed as described [35 (link)] with some modifications. Total RNA was isolated from MKN-7 cells using the RNeasy Minikit (Qiagen). The cDNA was synthesized using AMV reverse transcriptase (Promega) and a full-length human E-cadherin cDNA fragment containing NheI and XbaI sites was generated by PCR using PfxDNA polymerase (Invitrogen) and sense primer (5’-TATGCTAGCatgggcccttggagc-3’) and antisense primer (5’-TATtctagaaagtcgtcctcgccgcc-3’). The NheI/XbaI fragment of E-cadherin was inserted into a pcDNA3.1/myc-His(-)B vector (Invitrogen). Various extracellular domains of E-cadherin were deleted by inverse PCR using a KOD-Plus-mutagenesis kit (Toyobo) and primer sets as described [35 (link)]. The sequence of all expression vectors was confirmed by sequencing. The 293 cell line was transfected with the expression vectors using the Lipofectamine reagent described above and selected with G418. Cell extracts of cell membranes and non-membranes were prepared using ProteoExtract kit.