Cardiovascular Journal of Africa: Vol 27 No 1 (January/February 2016)

CARDIOVASCULAR JOURNAL OF AFRICA • Volume 27, No 1, January/February 2016

AFRICA

concentration determination,

to ensure equivalent amounts of

protein per sample were subjected to sodium dodecyl sulphate-

polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

Co-localisation

For co-localisation experiments, the differentiation media

and Esumi buffer was removed from the differentiated H9C2

rat-derived cardiomyocytes on the glass cover slips and the cells

were rinsed with PBS. The cells were permeabilised withmethanol

for five minutes at –20°C and fixed with 4% paraformaldehyde

for five minutes at room temperature. The cells were then washed

three times with PBS for 10 minutes and blocked in 1% BSA for

one hour at room temperature. Following the blocking step, the

cells were again washed three times with PBS for 10 minutes and

incubated at 4°C overnight with rabbit anti-KCNE2 (Abcam,

Biocom Biotech, RSA, 1:50) and goat anti-FLNC (Santa Cruz

Biotechnology Inc, USA, 1:50) primary antibodies diluted in

1% BSA.

The cells were then washed three times with PBS for 10

minutes and stained with Alexa 488 donkey anti-rabbit (Jackson

ImmunoResearch Laboratories Inc, USA, 1:500) and Cy3

donkey anti-goat (Jackson ImmunoResearch Laboratories Inc,

USA, 1:500) secondary antibodies in PBS for 90 minutes in the

dark at room temperature. Afterwards, the cells were washed

three times with PBS for 10 minutes, and Hoechst H-33342

was added for nuclear staining [Sigma-Aldrich (Pty) Ltd, RSA,

1:200; 10 mg/ml], followed by a 10-minute incubation at room

temperature.

Subsequently,thecoverslipswiththestainedcellsweremounted

onto glass slides using Mowiol (Jackson ImmunoResearch

Laboratories Inc, USA) containing

-propylgallate as the

anti-fade reagent and kept at 4°C in the dark until viewing.

Samples were acquired using the Carl Zeiss Confocal LSM

780 Elyra S1, equipped with a LSM780 GaAsP detector, using

a Plan Apochromat 63×/1.4 Oil DIC M27 or an alpha Plan-

Apochromat 100×/1.46 oil DIC objective (Central analytical

facility, Cell Imaging Unit, Stellenbosch University, RSA). The

samples were excited with a 488-nm and 561-nm laser under-

utilisation of a MBS 488/561 beam splitter.

Images were acquired through

-stacking with an increment

of 0.3-µm step width, and projected as maximum-intensity

projections using ZEN software (black edition, 2011). Thresholds

were determined using appropriate control images acquired for

cells individually stained (single-stain) for KCNE2 and FLNC,

respectively. The background was adjusted for all acquired

images using images of cells only stained with secondary control

antibodies.

Co-immunoprecipitation

Cells were harvested and the lysates were pre-cleared with

protein G agarose beads (KPL Inc, USA) for 30 minutes at 4°C.

The pre-cleared lysates (150 µg/total protein) were incubated

with 1 µg of either rabbit polyclonal anti-KCNE2

(Santa Cruz

Biotechnology Inc, USA) or goat polyclonal anti-FLNC (Santa

Cruz Biotechnology Inc, USA) antibody rotating overnight at

4°C.

To capture the protein complexes, 60 µl of protein G agarose

beads were added to the lysate and incubated for an additional

hour rotating at 4°C. The complexes were then washed three

times, each time removing the supernatant after centrifugation

and adding fresh lysis buffer that contained protease inhibitors

and PMSF. Proteins were eluted by addition of 1

SDS-PAGE

sample buffer [95%Laemmli sample buffer (Bio-RadLaboratories

Inc, USA), 5%

-mercapto-ethanol], denatured for five minutes

at 95°C and separated using 4–15% SDS-PAGE gels for Western

blot analysis. Two negative controls, a non-relevant antibody

control (HA-probe; Santa Cruz Biotechnology Inc, USA) and

a protein G agarose control (without antibody) were included in

all Co-IP experiments

Western blot analysis

Following co-IP, proteins were separated on 4–15% SDS-PAGE

gels and transferred to a polyvinylidene difluoride (PVDF)

membrane (Thermo Scientific, USA) by means of the iBlot

system (Invitrogen, USA). Membranes were blocked with

5% fat-free powdered milk, supplemented with Tris-buffered

saline Tween-20 (TBST, 0.01% Tween-20), for one hour at

room temperature. Membranes were then incubated at 4°C

overnight with the appropriate primary antibodies (Santa Cruz

Biotechnology Inc, USA, 1:200 anti-KCNE2; 1:1 000 anti-

FLNC), diluted with 5% milk in TBST.

Subsequently, the membranes were washed with TBST

and incubated for one hour at room temperature with the

corresponding horseradish peroxidase (HRP) conjugated

secondary antibodies (Santa Cruz Biotechnology Inc, USA,

1:2 000 donkey anti-rabbit; 1:2 000 donkey anti-goat), diluted

with 5% milk in TBST. Following incubation with the secondary

antibody, the membranes were washed for 30 minutes at room

temperature.

The SuperSignal

West Pico chemiluminescence substrate

kit (Thermo Scientific, USA) was then used according to the

manufacturer’s instructions and the membranes were exposed for

two minutes to CL-Xposure™ autoradiography film (Thermo

Scientific, USA). The autoradiography film was developed using

an Amersham hyperprocessor automatic autoradiography film

processor (Amersham Pharmacia Biotech UK Ltd, UK) prior

to final analyses.

Results

FLNC as a novel interactor with KCNE2 under

hypoxic conditions

The Y2H screen identified FLNC (GenBank: NP_001449.3) as

a KCNE2 (GenBank: NP_751951.1) interactor with the binding

regions located between amino acids 2637–2725 of FLNC and

amino acids 72–123 of KCNE2. These 88 amino acids of FLNC

are positioned at the end of the C-terminal domain, shown to be

involved in self-dimerisation.

Imaging analysis revealed a strong co-localisation signal

between KCNE2 and FLNC at the cell membrane, filamentous

structures and the cytoplasm of differentiated H9C2 rat-derived

cardiomyocytes under normoxic conditions (Fig. 1a–h), while the

co-localisation of these two proteins was mainly restricted to the

cytoplasmunder conditions of hypoxia (Fig. 1i–p). When the cells

were subjected to hypoxic stress, the pattern of co-localisation,

relative to that during normoxia, changed considerably at the

plasma membrane (Fig. 1i–p), where decreased co-localisation