Ab2811

The following antibodies were used for immunofluorescence (IF) and western blotting (WB): rat monoclonal anti-importin-α1 (1:100 for IF, 1:1000 for WB; 2G7; ab22534; Abcam), rat polyclonal anti-importin-α1 (1:100 for IF, 1:1000 for WB; I9658; Sigma-Aldrich), mouse monoclonal anti-importin-α (1:100 for IF, 1:1000 for WB; I1784; Sigma-Aldrich), mouse monoclonal anti-importin-β (1:100 for IF, 1:1000 for WB; 3E9; ab2811; Abcam), rabbit polyclonal anti-importin-β (raised against GST-tagged full-length importin-β and used at 1:1000 for WB), anti-histone H3 phospho-S10 (1:2000 for WB; 05-1336; Sigma-Aldrich), anti-α-tubulin (1:100 for IF, 1:2000 for WB; T9026; Sigma-Aldrich), anti-GFP (1:2000 for WB; sc-9996; Santa Cruz Biotechnology), anti-KIFC1 (in-house polyclonal, raised against his-tagged 887-end of KIFC1 and used at 1:100 for IF, 1:1000 for WB), rabbit polyclonal anti-TPX2 (1:100 for IF, 1:1000 for WB; 12245; Cell Signaling Technology), anti-NuMA (1:1000 for WB; ab109262; Abcam), anti-Ran (1:5000 for WB; 610340; BD Biosciences) and anti-Myc (1:1000 for WB; MABE282; Sigma-Aldrich). Kinase inhibitors were used at the following concentrations: 10 µM purvalanol A(Sigma-Aldrich), 9 µM Ro-3306 (Sigma-Aldrich), 0.1 µM BI2536 (Axon Medchem), 2 µM ZM447439(Selleck), 0.5 μM LY294009 (Selleck).

Cells were cultured on coverslips, which were treated by 10% goat serum for an hour at 37°C. Then, cells were treated by antibodies against KPNB1 (ab2811, Abcam Inc., dilution: 1:100), CNBP (sc‐515387, Santa Cruz Biotechnology, Inc., dilution: 1:100) or SMARCC2 (ab243634, Abcam Inc., dilution: 1:100) for 2 h. Coverslips were stained by 4′,6‐diamidino‐2‐phenylindole (300 nmol L⁻¹) after incubating with Alexa Fluor 488 (ab150081) or Alexa Fluor 594 (ab150160, Abcam Inc.) goat anti‐rabbit IgG.

Anti-GST (B-14) – mouse monoclonal – Santa Cruz Biotechnology, sc-138; Anti-HA (16B12) – mouse monoclonal – Covance, MMS-101P; Anti-MBP – mouse monoclonal – NEB, E8032S; Anti-Imp-β (3E9) – mouse monoclonal – abcam, ab2811; Anti-KPNA7 – rabbit polyclonal – Millipore Sigma, HPA031395; Anti-hnRNP R – rabbit polyclonal – Millipore Sigma, SAB2700924; Anti-hnRNP U (3G6) – mouse monoclonal – Santa Cruz Biotechnology, sc-32315; Anti-Flag M2 – mouse monoclonal – Millipore Sigma, F1804; Anti-CTCF – rabbit monoclonal – Cell Signaling, D31H2.

Immunoblotting was performed as described previously^{7 (link)}. Antibodies used in this study were as follows: NUP155 (A303-934A, Bethyl Laboratories), IPOβ (ab2811, Abcam), KNTC1 (F2051-10H4, Sigma-Aldrich), hCAP-D2 (E4161-4C12, Sigma-Aldrich), IPO7 (ab88339, Abcam), CLTC (sc-271178, Santa Cruz), Cdc2-phosphorylated vimentin Ser55 (D076-3, MBL), GST (G7781, Sigma-Aldrich), GFP (A6455, Invitrogen), and HA-tag (clone 3F10, Roche). Secondary antibodies were HRP-rabbit anti-mouse IgG (H + L) (61-6520, Invitrogen) or HRP-goat anti-rabbit IgG (H + L) (65–6120, Invitrogen).

After labelling with 100 nM LD655-tetrazine in TB as described above, the COS-7 cells were immediately fixed with 2% PFA in PBS for 10 min or kept at room temperature for 2 h and then fixed. After washing with PBS twice, the cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min. Then, the cells were washed with PBS twice and incubated with blocking buffer (3% BSA in PBS) for 90 min. The cells were subsequently immunolabelled with anti-KPNB1 antibody (ab2811, Abcam; at 1:1,000) and anti-mouse Alexa Fluor 488 secondary antibody (A-11001, Thermo Fisher; at 1:1,000). Immunofluorescence demonstrated that endogenous importin-β colocalized at the nuclear envelope after permeabilization and was retained during the 2-h time window of FLIM–FRET measurements (Extended Data Fig. 3e,f).

The following antibodies were used: MAb414 (ab24609, Abcam) (1:500 dilution), anti-RanGAP1 (ab92369, Abcam) (1:500), rabbit anti-lamin B1 (ab16048, Abcam) (1:500 dilution), anti-importin B1 (KPNB1) clone 3E9 (Abcam, ab2811) (1:500), anti-alpha tubulin (clone DM1A; T9026, Sigma) (1:5000), anti-CLASP2 (KT69, Absea) (1:100), anti-TaSP (a gift from Jabbar Ahmed [57 (link)]) (1:50,000), mouse monoclonal 1C12 (38) (1:500), rat anti-TaMISHIP (1:500) (15 (link)), rabbit anti-SuAT1 (a gift from Brian Shiels [12 (link)] [polyclonal R685]) (1:500), and mouse anti-PIM40.3 (ILRI Nairobi) (1:1,000). T. gondii was labeled with whole T. gondii extract rabbit antiserum (54 (link)) (1:1,000). The P. berghei parasitophorous vacuole membrane was labeled with rabbit anti-UIS4 antiserum (provided by P. Sinnis, Baltimore, MD, USA) (1:500).