Production of Cells with Targeted Integration of Gene Variants of Human ABC Transporter for Stable and Regulated Expression Using the Flp Recombinase System - Protein Misfolding and Cellular Stress in Disease and Aging - page 140

1. NucleoSpin ® RNA II kit (Macherey-Nagel GmbH & Co.
KG, Duren, Germany).
2. SuperScript II RT (Invitrogen, Carlsbad, CA, USA) using
random hexamers.
3. Thermal cycler (e.g., iCycler TM thermal cycler (BIO-RAD,
Hercules, CA, USA)).
4. Primer sets: ABCG2 (5′-GATCTCTCACCCTGGGGCT
TGTGGA-3′, 5′-TGTGCAACAGTGTGATGGCAAGGG
A-3′), GAPDH (5′-ACTGCCAACGTGTCAGTGGTGGA
CCTGA-3′, 5′-GGCTGGTGGTCCAGGGGTCTTACTCC
TT-3′).
5. TaqMan ® Fast Universal Master Mix (Applied Biosystems,
Foster City, CA, USA).
6. TaqMan ® probes (ABCG2; Hs00184979_m1, GAPDH;
Hs99999905_m1) (Applied Biosystems, Foster City, CA,
USA).
7. 7500 Fast Real Time-PCR System (Applied Biosystems,
Foster City, CA, USA).

2.2.2. qPCR Analysis

1. BXP-21 (ALEXIS Co., Lausen, Switzerland), a specific anti-
body to human ABCG2.
2. Anti-mouse IgG-horseradish peroxidase (HRP)-conjugate
(Cell Signaling Technology, Beverly, MA, USA).
3. Phosphate-buffered saline (PBS).
4. Mouse monoclonal antibody against GAPDH (American
Research Products, Inc., Belmont, MA, USA).
5. Lysis buffer, 50 mM Tris/HCl (pH 7.4), 1% (w/v) Triton
X-100, 1 mM DTT, supplemented with protease inhibitor
cocktail (Roche Ltd., Mannheim, Germany).
6. 27-gauge needle.
7. Endo H or PNGase F (New England Biosciences, Ipswich,
MA).
8. b-Mercaptoethanol (ME).
9. SDS-PAGE equipment with 7.5% (w/v) polyacrylamide
gels.
10. Hybond-ECL nitrocellulose membranes (GE Healthcare UK
Ltd., Buckinghamshire, UK).
11. Western Lighting Chemiluminescent Reagent Plus
(PerkinElmer Life And Analytical Sciences, Inc., Boston, MA,
USA).
12. Lumino Imaging Analyzer FAS-1000 (TOYOBO Co., Ltd.,
Osaka, Japan).

2.2.3. N-Linked
Glycosylation