Anti pa tag antibody

ChIP analysis was conducted as described [33 (link)]. Briefly, DNA was fragmented by sonication to an average size of 400–500 bp for standard ChIP or of 100–200 bp for high-resolution ChIP. Immunoprecipitation was conducted using Dynabeads Protein G (Invitrogen, Carlsbad, CA, USA), anti-FLAG-tag antibody (Sigma-Aldrich, St. Louis, MO, USA; M2), anti-PA-tag antibody (FujiFilm Wako Pure Chemical Corporation, Osaka, Japan; NZ-1), and polyclonal antibody against Rap1 (Santa Cruz Biotechnology, Dallas, TX, USA; yC-19) or Hmo1 [29 (link)]. Real-time quantitative PCR analyses were performed using a KAPA SYBR Fast qPCR kit (Kapa Biosystems, Wilmington, MA, USA). Each experiment was performed in triplicate, and the mean and standard deviation of the ratio of immunoprecipitated DNA to input DNA (IP/input) were calculated. Primer pairs used for quantitative PCR are described in the S1 text.

The AFM technique was used to visualize the immobilization of the CCT complex on the surface coated with anti-PA tag antibody, which was covalently immobilized on mica, following the procedure described in the literature [54 (link),55 (link)]. Briefly, 2 μL of 0.01% APTES (Sigma Aldrich, Burlington, MA, USA) diluted in ultra-pure water was applied to mica and incubated for 3 min, followed by two rounds of washing with ultra-pure water to remove any unbound APTES. Next, 0.2% glutaraldehyde was added to the APTES-modified mica and incubated for 5 min. After washing away the unbound glutaraldehyde with PBS, the anti-PA tag antibody (FUJIFILM Wako Chemicals, Osaka, Japan) was applied. Any unbound antibodies were removed, and free aldehyde groups were neutralized by washing with tris-buffered saline. Subsequently, the CCT complex was incubated in Buffer A for 10 min before proceeding to AFM observation. The custom-made high-speed AFM, equipped with the electro-beam deposition probe on the top of the ultra-short cantilever (BL-AC10DS-A2, Olympus, Tokyo, Japan), was operated following the procedure described in the literature [56 (link),57 (link),58 (link)].

The AFM technique was used to visualize the immobilization of the CCT complex on the surface coated with anti-PA tag antibody, which was covalently immobilized on mica, following the procedure described in the literature [54 (link),55 (link)]. Briefly, 2 μL of 0.01% APTES (Sigma Aldrich, Burlington, MA, USA) diluted in ultra-pure water was applied to mica and incubated for 3 min, followed by two rounds of washing with ultra-pure water to remove any unbound APTES. Next, 0.2% glutaraldehyde was added to the APTES-modified mica and incubated for 5 min. After washing away the unbound glutaraldehyde with PBS, the anti-PA tag antibody (FUJIFILM Wako Chemicals, Osaka, Japan) was applied. Any unbound antibodies were removed, and free aldehyde groups were neutralized by washing with tris-buffered saline. Subsequently, the CCT complex was incubated in Buffer A for 10 min before proceeding to AFM observation. The custom-made high-speed AFM, equipped with the electro-beam deposition probe on the top of the ultra-short cantilever (BL-AC10DS-A2, Olympus, Tokyo, Japan), was operated following the procedure described in the literature [56 (link),57 (link),58 (link)].