human abcb6  (Santa Cruz Biotechnology)

Journal: The Journal of Biological Chemistry

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1016/j.jbc.2023.105594

Figure Lengend Snippet: NanoBRET dilution assay reveals two putative heterodimers, ABCB5β/B6, and ABCB5β/B9. HEK-293T cells were transfected with decreasing amounts of NanoLuc and HaloTag constructs. Transfection started at 1 μg and gradually decreased to 0.125 μg per construct. DMSO refers to the technical negative control to which DMSO is added instead of ligand. Protein pairs serving as negative controls (ABCB2-ABCD1, ABCB3-ABCD1, ABCD1-ABCB5β) ( A ) and a positive control pair (ABCB2-ABCB3) ( B ) were tested alongside ABCB5β-ABCB6, ABCB5β-ABCB8, and ABCB5β-ABCB9 ( C ). Data are presented as mean ± SD. Simple linear regression was used to test whether the amount transfected significantly influenced the NanoBRET ratio ( n = 3). p -values are represented on graphs as ∗∗∗ p < 0.001, ∗∗ p < 0.01, and p > 0.05. For ABCB2-ABCD1 ( R 2 = 0.8719, F (1,10) = 68.07), ABCB3-ABCD1 ( R 2 = 0.6144, F (1,10) = 15.94), ABCB5β-ABCD1 ( R 2 = 0.6661, F (1,10) = 19.95), and ABCB5β-ABCB8 ( R 2 = 0.7526, F (1,10) = 30.41), the relation between both parameters was statistically significant. For ABCB2-ABCB3 ( R 2 = 0.0269, F (1,10) = 0.2767), ABCB5β-ABCB6 ( R 2 = 0.0011, F (1,10) = 0.0113), and ABCB5β-ABCB9 ( R 2 = 0.0204, F (1,10) = 0.2080), changes in the amount of transfected DNA did not influence the NanoBRET ratio. DMSO, dimethyl sulfoxyde; NanoBRET, nanoluciferase-based bioluminescence resonance energy transfer.

Article Snippet: Four hours later, transfection with jetPRIME (Polyplus transfection) was carried out following the manufacturer’s instructions. shRNAs constructs for the stable knockdown of human ABCB6 (sc-94721-SH) and ABCB9 (sc-60115-SH) were obtained from Santa Cruz Biotechnology.

Techniques: Dilution Assay, Transfection, Construct, Negative Control, Positive Control, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1016/j.jbc.2023.105594

Figure Lengend Snippet: Saturation assay confirms specific ABCB5β–ABCB6 and ABCB5β–ABCB9 interactions. A , the concentration of the donor is held constant while the concentration of the acceptor is increased. If the interaction is specific, the signal will reach a plateau where all donors are saturated with acceptors, and luminescence will no longer increase. In the case of a nonspecific interaction, the signal will continue to increase linearly. For each square, the schematic gray halo represents the intensity of the NanoBRET signal observed from low to high. In graphs ( B and C ), the percent of the maximum NanoBRET ratio is plotted against the NanoLuc−HaloTag ratio. B , donor saturation assay for the positive control, ABCB2-ABCB3, and the negative controls, ABCB2-ABCD1, ABCB3-ABCD1, and ABCB5β-ABCD1. C , donor saturation assay for ABCB5β-ABCB6, ABCB5β-ABCB8, and ABCB5β-ABCB9. NanoBRET, nanoluciferase-based bioluminescence resonance energy transfer.

Article Snippet: Four hours later, transfection with jetPRIME (Polyplus transfection) was carried out following the manufacturer’s instructions. shRNAs constructs for the stable knockdown of human ABCB6 (sc-94721-SH) and ABCB9 (sc-60115-SH) were obtained from Santa Cruz Biotechnology.

Techniques: Saturation Assay, Concentration Assay, Positive Control, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1016/j.jbc.2023.105594

Figure Lengend Snippet: Coimmunoprecipitation of ABCB5β-ABCB6 and ABCB5β-ABCB9 demonstrates the presence of these heterodimers in Mel JuSo and UACC-257 cells. The Mel JuSo ( A ) or UACC-257 ( B ) proteins were immunoprecipitated (IP) with either an anti-ABCB5, anti-ABCB6, or anti-ABCB9 antibody. The precipitated proteins were revealed by Western blotting after SDS-PAGE using the corresponding antibody indicated on the right side of each blot. Fifteen micrograms of total proteins from the starting cell lysate were loaded in the first lane, while the total IP eluate was loaded on the gel. ABCB5β was expressed in High-Five insect cells, and total membrane proteins were prepared and loaded on the gel (High5 ABCB5β) as a complementary molecular weight marker when using anti-ABCB5. An isotype control was performed to determine the specificity of the signal obtained in Western blot.

Article Snippet: Four hours later, transfection with jetPRIME (Polyplus transfection) was carried out following the manufacturer’s instructions. shRNAs constructs for the stable knockdown of human ABCB6 (sc-94721-SH) and ABCB9 (sc-60115-SH) were obtained from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot, SDS Page, Membrane, Molecular Weight, Marker, Control

Journal: The Journal of Biological Chemistry

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1016/j.jbc.2023.105594

Figure Lengend Snippet: Proximity ligation assay confirms the interactions between ABCB5β-ABCB6 and ABCB5β-ABCB9 protein pairs in Mel JuSo and UACC-257. A , confocal images of PLA in Mel JuSo and UACC-257 that stably expressed either a nontargeting shRNA or an ABCB6-specific (or ABCB9-specific) shRNA. The detected PLA signal is in red . Nuclei are stained in blue using DAPI. The scale bar represents 40 μM. After shRNA knockdown, efficiency was determined using reverse transcription-quantitative polymerase chain reaction and Western blots in Mel JuSo ( B ) and UACC-257 ( C ). 18S and tubulin were used as references for reverse transcription-quantitative polymerase chain reaction and Western blots normalization, respectively (n = 2). ImageJ ( https://imagej.net/ij/download.html ) was used to quantify protein expression in the Western blot as well as the average number of dots per cell ( n = 3) ( D ). Scramble and shRNA quantifications are shown along with technical negative controls, including PLA conducted using either one antibody (AB) or none. Data is presented as mean ± SD. In Mel JuSo: ABCB6 scramble (39.89 ± 14.21), ABCB6 shRNA (0.28 ± 0.60), ABCB5 AB control (0.55 ± 0.91), ABCB6 AB control (0 ± 0), ABCB9 scramble (16.89 ± 4.27), ABCB9 shRNA (0.84 ± 1.04), ABCB9 AB control (0 ± 0.01), and no antibody control (0.01 ± 0.05). In UACC-257: ABCB6 scramble (42.16 ± 10.62), ABCB6 shRNA (0.97 ± 1.00), ABCB5 AB control (0.01 ± 0.02), ABCB6 AB control (0.03 ± 0.07), ABCB9 scramble (25.39 ± 6.74), ABCB9 shRNA (1.04 ± 1.16), ABCB9 AB control (0.79 ± 0.58), and no antibody control (0 ± 0). Student’s t test was performed between the scramble and shRNA conditions. Statistical significances are presented on graph as ∗∗∗∗ p < 0.0001. ABCB5β/B6 in Mel JuSo ( t = 9.648, df = 22), ABCB5β/B9 in Mel JuSo ( t = 12,66, df = 22), ABCB5β/B6 in UACC-257 ( t = 13,38, df = 22), ABCB5β/B9 in UACC-257 ( t = 12,34, df = 22). DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Four hours later, transfection with jetPRIME (Polyplus transfection) was carried out following the manufacturer’s instructions. shRNAs constructs for the stable knockdown of human ABCB6 (sc-94721-SH) and ABCB9 (sc-60115-SH) were obtained from Santa Cruz Biotechnology.

Techniques: Proximity Ligation Assay, Stable Transfection, shRNA, Staining, Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1016/j.jbc.2023.105594

Figure Lengend Snippet: Expression of ABCB5β, ABCB6, ABCB5β_P-gp linker_ABCB6, ABCB6_P-gp linker_ABCB5β, ABCB9, ABCB5β_P-gp linker_ABCB9, and ABCB9_P-gp linker_ABCB5β in High-Five insect cells. Total membranes vesicles prepared from High-Five cells infected with baculovirus containing either ABCB5β, ABCB6, ABCB9, ABCB5β_P-gp linker_ABCB6, ABCB6_P-gp linker_ABCB5β, ABCB5β_P-gp linker_ABCB9, ABCB9_P-gp linker_ABCB5β and their corresponding EQ mutants were subjected to SDS-PAGE, followed by Coomassie-blue staining ( A ) or Western blotting with anti-ABCB5, anti-ABCB6, and anti-ABCB9 antibodies ( B ). C , Western blotting with an anti-ABCB1 antibody was performed to assess possible cross reactivity with ABCB5β, ABCB6, and ABCB9 and their chimeric heterodimers. Bands of interest are highlighted by black arrows .

Article Snippet: Four hours later, transfection with jetPRIME (Polyplus transfection) was carried out following the manufacturer’s instructions. shRNAs constructs for the stable knockdown of human ABCB6 (sc-94721-SH) and ABCB9 (sc-60115-SH) were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Infection, SDS Page, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1016/j.jbc.2023.105594

Figure Lengend Snippet: Expression of homodimers in High-Five cells and BeFx-sensitive ATPase activity. A , two-dimensional schematic representation of ABCB5β ( purple ), ABCB6 ( orange ), and ABCB9 ( gray ) structure based on CCTOP predictions . ABCB5β has one complete and another partial NBD and one TMD comprised of six transmembrane helices (TMs). The N-terminal NBD (NBD1) is truncated and lacks the conserved Walker A domain. ABCB6 has six transmembrane helices (TM6-TM11) that constitute its TMD1 and five additional transmembrane helices (TM1-TM5) in the N terminus that form the TMD0. ABCB9 has a TMD1 consisting of six transmembrane helices (TM5-10) and a TMD0 made of four transmembrane helices at the N terminus. The location of each EQ mutation is represented by red Xs. B , pixel intensity quantification of the protein bands of interest, after Coomassie-blue staining was performed using ImageJ (n = 3). C , ABCB5β, ABCB6, ABCB9, and their corresponding EQ mutants’ ATPase activities were measured in the presence and absence of BeFx as described in the methods section. On the left , data are shown as ATPase activity in the absence of BeFx (total ATPase activity) and in the presence of BeFx (BeFx-sensitive ATPase activity). On the right , data are represented by subtracting BeFx-resistant ATPase activity from the total ATPase activity (n = 3). In all panels, the names of EQ mutants are shown in red . NBD, nucleotide-binding domain; BeFx, beryllium fluoride.

Article Snippet: Four hours later, transfection with jetPRIME (Polyplus transfection) was carried out following the manufacturer’s instructions. shRNAs constructs for the stable knockdown of human ABCB6 (sc-94721-SH) and ABCB9 (sc-60115-SH) were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Activity Assay, Mutagenesis, Staining, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1016/j.jbc.2023.105594

Figure Lengend Snippet: Expression level and BeFx-sensitive ATPase activity of heterodimers. A , two-dimensional schematic representation of the ABCB5β/B6 and ABCB5β/B9 heterodimers after fusion with the P-gp flexible linker based on CCTOP predictions . The P-gp linker is shown in green and highlighted with a green arrow ; ABCB5β is represented in purple , ABCB6 in orange , and ABCB9 in gray . The ABCB5β_P-gp linker_ABCB6 has 16 transmembrane helices. The ABCB6_P-gp linker_ABCB5β has 17 transmembrane helices and the ABCB5β_P-gp linker_ABCB9 and ABCB9_P-gp linker_ABCB5β both have 16 transmembrane helices. The location of EQ mutations is represented by red Xs. B , the Coomassie-blue stained gel pixel intensity of the bands of interest were quantified using ImageJ (n = 3). C , the graph on the left shows the ATPase activity for all heterodimer chimeras and their corresponding EQ mutants measured in both the absence and presence of BeFx as described in the method section, total ATPase activity, and BeFx-sensitive ATPase activity, respectively. On the right , data represent BeFx-sensitive ATPase activity, which is calculated by subtracting BeFx ATPase activity from the total ATPase activity (n = 3). In all panels, the names of EQ mutants are shown in red . BeFx, beryllium fluoride.

Article Snippet: Four hours later, transfection with jetPRIME (Polyplus transfection) was carried out following the manufacturer’s instructions. shRNAs constructs for the stable knockdown of human ABCB6 (sc-94721-SH) and ABCB9 (sc-60115-SH) were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Activity Assay, Staining