MIN6B1-SNAP_GLP-1R
cells
were incubated in HEPES-bicarbonate buffer supplemented with 3 mMd -glucose for 2 h at 37 °C and either fixed in 4% paraformaldehyde
or further stimulated with 11 mMd -glucose (G11) containing
10 nM Ex-4(1–39) or 800 nM ExONatide for 1 h at 37 °C.
Cells were either fixed or washed extensively prior to incubation
with 10 μM Ex-4(9–39) for a further 1 or 3 h following
0 or 10 min application of 10 mM BME. At the concentration used here,
Ex4(9–39) is expected to effectively trap GLP-1R at the plasma
membrane, allowing recycling to be quantified via reappearance of
labeled receptor. Control experiments were performed using BME and
Ex4(1–39)-alone. Immunostaining was performed using mouse antihuman
GLP-1R (1:30; Mab 3F52; Developmental Studies Hybridoma Bank) and
rabbit anti-EEA1 antibodies (1:50; sc-33585; Santa Cruz Biotechnology),
before secondary antimouse AlexaFluor-488 staining and mounting on
coverslips with Vectashield Hardset + DAPI. Images were captured using
a Zeiss LSM780 microscope and a 63x/1.4 NA oil objective.
Excitation was delivered at λ = 405 nm and λ = 481 nm.
Emitted signals were collected at λ = 410–495 nm and
λ = 493–630 nm for DAPI and AlexaFlour-488, respectively.
Surface signal was quantified on binarized images using the threshold
plugin for ImageJ and expressed relative to the total signal.
cells
were incubated in HEPES-bicarbonate buffer supplemented with 3 mM
or further stimulated with 11 mM
10 nM Ex-4(1–39) or 800 nM ExONatide for 1 h at 37 °C.
Cells were either fixed or washed extensively prior to incubation
with 10 μM Ex-4(9–39) for a further 1 or 3 h following
0 or 10 min application of 10 mM BME. At the concentration used here,
Ex4(9–39) is expected to effectively trap GLP-1R at the plasma
membrane, allowing recycling to be quantified via reappearance of
labeled receptor. Control experiments were performed using BME and
Ex4(1–39)-alone. Immunostaining was performed using mouse antihuman
GLP-1R (1:30; Mab 3F52; Developmental Studies Hybridoma Bank) and
rabbit anti-EEA1 antibodies (1:50; sc-33585; Santa Cruz Biotechnology),
before secondary antimouse AlexaFluor-488 staining and mounting on
coverslips with Vectashield Hardset + DAPI. Images were captured using
a Zeiss LSM780 microscope and a 63x/1.4 NA oil objective.
Excitation was delivered at λ = 405 nm and λ = 481 nm.
Emitted signals were collected at λ = 410–495 nm and
λ = 493–630 nm for DAPI and AlexaFlour-488, respectively.
Surface signal was quantified on binarized images using the threshold
plugin for ImageJ and expressed relative to the total signal.