Anti phf5a

For immunoblotting of SF3B1/FLAG-SF3B1proteins in transiently-transfected HEK293T and stably-transduced TF1 and K562 cells in
which transcriptome analysis was done in parallel, Western blotting using a mouse
anti-human-SF3B1 antibody (Abcam #172634) at 1:1000 dilution was used.
Immunoblotting following affi nity purification of SF3B1 in HEK293T and TF1 cells was
performed as previously described, and primary antibodies were: anti-SF3B1 (Bethyl
Laboratories, A300–996A, 1:1,000), anti-ACTIN (Sigma, A2066, 1:2,000),
anti-DYKDDDDK (GenScript, A00187, 1:1,000), anti-SUGP1 (Bethyl
Laboratories A304–675A-M, 1:1,000), and anti-PHF5A (Proteintech 15554–1-AP,
1:1000)^{17 (link)}. Secondary antibodies
were: Donkey anti-Rabbit IgG (LI-COR, 926–68073, 1:5,000) and Goat anti-Mouse IgG
(LI-COR, 926–32210, 1:5,000).

All animal experiments were performed according to institutional guidelines. For xenograft assays, H1299 cells (1 × 10⁷) were resuspended in 200 μl serum-free RPMI 1640 and Matrigel (BD Biosciences; 1:1), and implanted subcutaneously into the flanks of 4-week old BALB/c nu/nu female nude mice. The mice were monitored every 3 days; tumor length and width measurements were performed with calipers, and tumor volumes were derived as length×width² × 0.5 (mm³). At 30 days, tumors were detected by an IVIS imaging system, excised, weighted, and paraffin-embedded following necropsy. Serial 5.0 μm sections were obtained and assessed by IHC using anti-PHF5A and anti-Ki67 antibodies (Proteintech). The proliferation index was determined as the proportion of Ki67-positive cells.