Journal of Shanghai Normal University Natural Sciences Feb SNAT2. HEK293T Western blot SNAT2 - HA Q 31 A

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1 4 1 1 Vol 41 No Journal of Shanghai Normal University Natural Sciences Feb SNAT2 - HA * SNAT2 - SNAT2 PCR HA SNAT2 C pbk - CMVΔ SNAT2 - HA HEK293T Western blot SNAT2 - HA pbk - CMVΔ SNAT2 - HA SNAT2 SNAT2 Q 31 A Sodium - coupled Neutral Amino Acid Transporter SNAT SLC38 SLC38 System A System N System A SNAT1 SNAT2 SNAT4 alanine System N SNAT3 SNAT5 glutamine 1 SNAT2 System A SNAT kda 11 TMD SNAT2 1 1 ph SNAT2 1 SNAT2 SNAT2 α - 2 SNAT2 SNAT2 SNAT2 6 TMD1 82 TMD ZZ1039 S30406 DZL *

2 SNAT2 SNAT2 SNAT2 3 SNAT2 SNAT SNAT SNAT2 PCR SNAT2 - HA pbk - CMVΔ SNAT2 - HA human embryonic kidney cell HEK293T cell SNAT pbk - CMVΔ pbk - CMVΔ SNAT2 E coli DH5α Tiangen HEK293T PCR taq mix Generay DNA marker 1kb Spectra TM Multicolor High Range Protein Ladder Fermentas Wizard Plus SV Minipreps DNA Purification System Promega Gel Midi Purification Kit TIANGEN Agarose BIOWEST XbaI BlpI T4 DNA ligase CIP NEB DMEM High Glucose1 0 25% Trypsin - EDTA1 Gibco Lipofectamine TM 2000 Invitrogen BCA Protein Assay Kit Pierce cocktail Roche PBS Tris TEMED N N - PVDF ECL Millipore X - OMAT BT X Kodak Mouse Anti - c - HA Tag Monoclonal Antibody DTT SDS Goat Anti - Mouse IgG H&L - HRP Conjugated Secondary Antibody Sigma pbk - CMVΔ - SNAT2 XbaI HA - tag BlpI Forward primer 5' - GCT /CTAG /A XbaI GATCCCTCGAC- CTCGAGATC - 3' Reverse primer 5' - CGCGGGC /TAA /GC BlpI TTAGGCATAATCGGGTACATCGTA- AGGGTA HA ATGTCCGCCTGCAGAGGCATCGTG - 3' 2 2 PCR pbk - CMVΔ - SNAT2 PCR 0 5 ng /μl 0 4μmol /L PCR 94 5 min s s 72 1 min min 4 1% PCR 2 3 pbk - CMVΔ - SNAT2 - HA XbaI BlpI PCR 3 h 1% pbk - CMVΔ SNAT2 3 h 30 min 1% Agarose h E coli DH5α

3 1 SNAT2 - HA PCR PCR Kan r LB Kan r LB PCR F - 5' - CTTGGGGTATACCAGCGGCCTTTCTC - 3' R - 5' - ATCTCCATTCGCCATTCAGG - 3' PCR 2603bp PCR Taq Master Mix 2 5 μl BstZ17I - F 0 4 μmol /L DXY - R 0 4 μmol L μl ddh 2 O 10 μl PCR 94 2 min s s 72 1 min min 4 pbk - CMVΔ - SNAT2 - HA XbaI BlpI 10 NEB buffer 41 μl 100 BSA 0 1 μl XbaI 20U 0 1 μl BlpI 20U 0 1 μl 400 ng ddh 2 O 10 μl 37 1 h XbaI BlpI SNAT2 - HA 2 5 SNAT2 - HA HEK293T 10 cm 5% CO h 90% Lipo μg Lipo μg /μl DMEM - 5 min 300 μl HEK293T 4 ~ 6 h 5% CO h 10 pbk - CMVΔ - SNAT2 HEK293T pbk - CMVΔ - SNAT2 - HA HEK293T h PBS 0 1 mmol /L Ca mmol /L Mg min 3 1 ml RIPA 50 mmol /L Tris - HCl ph mmol /L NaCl 1 mmol /L EDTA 1% TritonX % 0 1% SDS cocktail eppendorf 30 min r /min 15 min 200 μl BCA r /min 30 min 100 μl 40 mmol /L Tris - HCl ph7 4 5 mmol /L MgCl mmol /L EGTA BCA 1 / Western Blot Western Blot SNAT2 1 10% SDS - PAGE 10 μg 2 μg 2 PVDF 4 40 V 2 h 3 PVDF TBST 5% 1 ~ 2 h 4 TBST 2 5% HA h TBST 15 min 3 IgG - HRP h TBST 15 min pbk - CMVΔ - SNAT2 PCR PCR 5 μl 1% bp bp PCR XbaI BlpI bp 1 A pbk - CMVΔ - SNAT2 XbaI BlpI 3 XbaI - XbaI - BlpI XbaI BlpI 3 XbaI - XbaI bp XbaI - BlpI 202 bp BlpI - XbaI bp 1 B BlpI - XbaI bp

4 E coli DH5α Kan r 7 PCR XbaI BlpI 5825 bp 1890 bp 2 A 1 1 kb DNA marker 2 PCR 1890 bp B 1 1 kb DNA marker 2 pbk - CMVΔ SNAT2 BlpI - XbaI 5825 bp XbaI - XbaI 2986 bp XbaI - BlpI 202 bp BlpI - XbaI 5825 bp 1 1 1kb DNA marker bp bp 2 DNA pbk - CMVΔ - SNAT2 - HA Western Blot SNAT2 - HA pbk - CMVΔ - SNAT2 - HA HEK293T 36 h 10% SDS - PAGE HA SNAT2 pbk - CMVΔ - SNAT2 4 SNAT2 - HA 50 ~ 70 k SNAT2 56 kda SNAT2 5 2 pbk - CMVΔ - SNAT2 - HA 1 pbk - CMVΔ - SNAT2 3 pbk - CMVΔ - SNAT2 - HA HEK293T 10 μg 2 pbk - CMVΔ - SNAT2 HKE293T 10 μg 3 pbk - CMVΔ - SNAT2 - HA HEK293T 2 μg 4 pbk - CMVΔ - SNAT2 - HA HEK293T 10 μg 4 Western Blot

5 1 SNAT2 - HA 87 SNAT2 11 N C SLC38 SNAT2 5 SNAT2 4 SNAT2 - HA pbk - CMV CMV SNAT2 HEK293T HA 9 SNAT2 Sodium - coupled Neutral Amino Acid Transporter 2 SNAT2 IL - 6 SNAT2 7 SNAT2 SNAT2 SNAT2 11 SNAT2 4 PCR HA SNAT2 C PCR 1 2 pbk - CMVΔ - SNAT2 - HA 3 HEK293T Western Blot SNAT2 - HA 56 kd 4 SNAT2 - HA HA SNAT2 SNAT2 1 MACKENZIE B ERICKSON J D Sodium-coupled neutral amino acid System N /A transporters of the SLC38 gene family J PflugersArch-Eur J Physio YAO D MACKENZIE B MING H et al Novel System A Isoform Mediating Na1 /Neutral Amino Acid Cotransport J JBC J

6 M GUO Z Y CHANG C C LU X et al The disulfide linkage and the free sulfhydryl accessibility of acyl-coenzyme a cholesterol acyltransferase 1 as studied by using mpeg - maleimide J Biochemistry ZHANG Z GREWER C The Sodium-Coupled Neutral Amino Acid Transporter SNAT2 Mediates an Anion Leak Conductance that Is Differentially Inhibited by Transported Substrates J Biophysical Journal JONES H N JANSSON T POWELL T L IL - 6 stimulates system a amino acid transporter activity in trophoblast cells through STAT3 and increased expression of SNAT2 activity J Am J Physiol Cell Physiol ZHANG Z GAMEIRO A CROWER C Highly conserved asparagine 82 controls the interaction of Na + coupled neutral amino acid transporter SNAT2 J JBC with the sodium 9 μ pires2 - EGFP HEK293 J Life Science Research Construction and characterization of Sodium coupled Neutral Amino acid Transporter SNAT2 HA fusion protein DONG Xiao-yun WANG Han MENG Wen LI Yang ZHANG Zhou * College of Life and Environment Sciences Shanghai Normal University Shanghai China Abstract Sodium coupled Neutral Amino acid Transporter 2 SNAT2 widespread expressed in the mammalian tissues plays an important role in the pathways of glutamate glutamine cycle and gluconeogenesis To determine easily SNAT2 expression and location in the membrane we construct an eukaryotic expression plasmid with HA at the C terminus of SNAT2 in pbk CMVΔ by molecular biological methods After transiently transfected into human embryonic kidney cells HEK293T correctly expression and location of SNAT2 HA fusion proteins are observed in cell membranes by western blot The plasmid pbk CMVΔ SNAT2 HA provides a good tool for studying structure and function of SNAT2 in the future Key words sodium - coupled neutral amino acid transporter 2 SNAT2 fusion protein construction and characterization