Anti hp1α

The following primary antibodies were used in this study: anti-H3K9me3 (Abcam, cat# ab8898; IF 1:1000, WB 1:2000), anti-H3K9me2 (Active Motif, cat# 39239, IF 1:1000), anti-Lamin A/C (Santa Cruz, cat# sc-376248; IF 1:500), anti-Lamin B1 (Abcam, cat# ab16048; IF 1:1000), anti-GFP (Abcam, cat# ab290; WB 1:1000, IP 1:200), Normal Rabbit IgG (Cell Signaling, cat# 2729; IP 1:200), anti-HP1α (Abcam, cat# 77256; IF, WB 1:1000), anti-HP1β (Abcam, cat# ab10478; IF, WB 1:1000), anti-HP1γ (Santa Cruz, cat# sc-398562; IF 1:500, WB 1:1000), anti-PRR14 (Proteintech, cat# 22819-1-AP; WB 1:1000), anti-β-Actin (8H10D10) (Cell Signaling, cat# 3700S; WB 1:5000), anti-Histone H3 (Abcam, cat# ab1791; WB 1:10,000), and anti-GAPDH (D4C6R) (Cell Signaling, cat# 51332; WB 1:1000).
The following secondary antibodies from ThermoFisher Scientific/Invitrogen were used: Donkey anti-Rabbit Alexa Fluor 568 (cat# A10042, IF 1:1000), Donkey anti-Mouse Alexa Fluor 488 (cat# A10037, IF 1:1000), Donkey anti-Rabbit Alexa Fluor 647 (cat# A31573, IF 1:1000), Donkey anti-Mouse Alexa Fluor 647 (cat# A31571, IF 1:1000); and HRP-linked anti-Rabbit IgG and HRP-linked anti-Mouse IgG (Cell Signaling, cat# 7074 and 7076; WB 1:5,000). IF – immunofluorescence, WB – Western Blot, IP – immunoprecipitation.

Preparation of chromosome spreads and subsequent fluorescence in situ hybridization (FISH) was described previously [46 (link)], except that we enriched for mitotic cells by treatment with 300 nM TN-16 (FUJIFILM Wako Pure Chemical Corp., Doshomachi Osaka, Japan) for 2 h. An evaluation of HAC formation was performed, as described previously [33 (link)].
The ChIP and subsequent qPCR and competitive PCR were described previously [45 (link)]. Anti-HP1α was purchased from abcam (Cambridge, UK; ab77256). PCR primers were from the literature [40 (link)], except N11F5 (5′-GGGATCACTAGCAATAAAAGGTAGAC-3′) and N11R6 (5′-TCCTTCTGTCTCGTTTTTATGGC-3′) used for competitive PCR of tetO vs. lacO, and N11F8 (5′-AGACAGAAGCATGCTCAGAAAC-3′) and N11R11 (5′-CTACCTTTTATTGCTAGTGATCCC-3′) for CENP-B box wild-type vs. mutant.

The total proteins of both cell lines were extracted and measured by Western blot analysis with the following antibodies: anti-H3K9me3 (Abcam, MA, USA; 1:1000), anti-HP1α (Abcam, MA, USA; 1:2000), anti-Ac-H3 (CST, 1:1000) and anti-β-Tubulin (Beyotime Biotechnology, 1:1000). The cells were washed triply with ice-cold PBS and treated with RIPA lysis buffer with 100 mM phenylmethanesulfonyl fluoride (PMSF) (Beyotime). An equal amount of total protein was subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA) then probed with primary antibodies overnight at 4°C. The membranes were then incubated for 1 h with the secondary HRP-conjugated antibodies (1:1000; Cell Signaling Technology). β-Tubulin was used for the loading control. The protein bands were visualized using the ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA) and their densities were measured using the Quantity One software (Bio-Rad Laboratories).