1 Materials and Methods FRAP microscopy: Images were acquired on a Leica TCS SP confocal microscope with a Leica PL APO 100x/1.4 oil immersion lens. For GFP excitation we used the 488 nm line of an Argon Krypton laser and fluorescence emission was collected between 500 to 560 nm. All experiments were done at 37°C. For the FRAP experiments a pre-bleach image was acquired by averaging four consecutive images. Then a single spot on the cell was bleached with a laser pulse, lasting between 0.1 and 0.5 s at 100% power without scanning. The objective lens used produced a laser spot of approximately 0.2 µm on the cell, but the bleached area was significantly larger, typically in the order of 1 to 2 µm. Single section images were then collected at 5-s intervals. For this part of the imaging the laser power was typically attenuated to 10% of the maximum. Fluorescence Recovery After Photobleaching (FRAP): Plots were generated from background subtracted images using the public domain software ImageJ (http://rsb.info.nih.gov/ij) following the analysis described in (1). In brief the signal I was measured in an area of interest (AOI) and normalised to the change in the total fluorescence due to the bleach pulse and the imaging: I = (To/Io)* It/Tt; where To and Tt is the total cellular fluorescence in the pre-bleach and post-bleach images, respectively, and Io is the average intensity in the AOI in the pre-bleach image.). Double staining microscopy: GFP and DAPI co-localization images were acquired using a Deltavision system (Applied Precision, USA) and a cooled CCD camera (Photometrics CH350L) on an Olympus IX70 inverted microscope, using a 100x/1.35 UPlanApo objective lens.
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Materials and Methods - Science...1 Materials and Methods FRAP microscopy: Images were acquired on a Leica TCS SP confocal microscope with a Leica PL APO 100x/1.4 oil immersion lens.
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1
Materials and Methods
FRAP microscopy: Images were acquired on a Leica TCS SP confocal microscope with a
Leica PL APO 100x/1.4 oil immersion lens. For GFP excitation we used the 488 nm line
of an Argon Krypton laser and fluorescence emission was collected between 500 to 560
nm. All experiments were done at 37°C. For the FRAP experiments a pre-bleach image
was acquired by averaging four consecutive images. Then a single spot on the cell was
bleached with a laser pulse, lasting between 0.1 and 0.5 s at 100% power without
scanning. The objective lens used produced a laser spot of approximately 0.2 µm on the
cell, but the bleached area was significantly larger, typically in the order of 1 to 2 µm.
Single section images were then collected at 5-s intervals. For this part of the imaging the
laser power was typically attenuated to 10% of the maximum.
Fluorescence Recovery After Photobleaching (FRAP): Plots were generated from
background subtracted images using the public domain software ImageJ
(http://rsb.info.nih.gov/ij) following the analysis described in (1). In brief the signal I was
measured in an area of interest (AOI) and normalised to the change in the total
fluorescence due to the bleach pulse and the imaging: I = (To/Io)* It/Tt; where To and Tt
is the total cellular fluorescence in the pre-bleach and post-bleach images, respectively,
and Io is the average intensity in the AOI in the pre-bleach image.).
Double staining microscopy: GFP and DAPI co-localization images were acquired using
a Deltavision system (Applied Precision, USA) and a cooled CCD camera (Photometrics
CH350L) on an Olympus IX70 inverted microscope, using a 100x/1.35 UPlanApo
objective lens.
2
Figure S1. The GFP tag does not interfere with HP1β chromatin binding in transgenic T
cells and does not affect binding to methylated histone H3 peptides in vitro. (A) T cells
were isolated from a transgenic mouse expressing GFP-HP1β or a non-transgenic control
mouse. Lysates were prepared from whole cells (WC) or cells sequentially extracted with
hypotonic buffer (CE), nuclear extraction buffer lacking (NE 0) or containing 400 mM
NaCl (NE400). The residual chromatin pellet (NE P) was also analyzed after addition of
SDS-PAGE sample buffer and sonication. Volumes of each fraction derived from the
same number of cells were analysed by Western blotting using a HP1β monoclonal
antibody that detects both HP1β and GFP-HP1β (lane M). Asterisk indicates a protein
that cross-reacts with the HP1β antibody and is found in both transgenic and control
mouse T cells. (B) Recombinant HP1β and GFP-HP1β were purified from E. coli as
HIS6-tagged fusion proteins and tested for their ability to bind to beads containing short
peptides derived from the N-terminal domain of histone H3. The peptides were
dimethylated (me2), trimethylated (me3) or acetylated (Ac) at either lysine-9 (K9) or
lysine-27 (K27). The amount of protein remaining bound to the H3 peptide beads after
extensive washes in binding buffer is shown alongside the equivalent of 100% of the
input protein (lanes labeled I). Lanes HM and LM contain protein size markers.
Figure S2. HP1α co-localizes with GFP-HP1β and DAPI dense heterochromatic foci in
transgenic T cells. Transgenic peripheral lymphocytes were fixed in paraformaldehye and
stained with anti-HP1α antibody (HP-1H5 mouse monoclonal IgG1 from P.