CRLF3 deficiency causes microtubule hyperstability. (A-H) Washed platelets maintained at 37°C (control [WT]) (A,B) (Crlf3^−/−) (C,D) or stored at 4°C for 3 hours (control [WT]) (E,F) (Crlf3^−/−) (G,H) adhered to poly-L-lysine–coated coverslips and stained for α-tubulin (green) and F-actin (red). Scale bars are 20 μm (A,C,E,G) and 5 μm (B,D,F,H). (I) Platelets retaining microtubule structures after incubation at 4°C were determined by manual counting of images for control (WT; blue) and Crlf3^−/− (red) mice (n = 3). (J-K) Representative western blots of in vitro cultured MK (J) or platelet (K) lysates against tyrosine α-tubulin, α-tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; left); acetylated α-tubulin, α-tubulin, and GAPDH (middle); or glutamylated α-tubulin (AG-20B-0020_upper band), α-tubulin, and GAPDH (right) for control (WT; blue) and Crlf3^−/− (red) samples. The quantification of glutamylated α-tubulin/total tubulin in in vitro cultured MKs (bar graphs on right) was carried out on 8 control and 8 Crlf3^−/− samples with 2 technical replicates. Where α-tubulin and GAPDH panels are the same for multiple tubulin modifications, membranes were stripped and reprobed between antibodies against specific tubulin modifications before finally being stripped and reprobed for α-tubulin and GAPDH. (L) In vitro cultured MKs were seeded onto fibrinogen-coated coverslips and incubated at 37°C for 5 hours to induce proplatelet formation. Samples were fixed, stained for polyglutamylated α-tubulin (AG-20B-0020), and imaged by fluorescence microscopy. Images are representative for Crlf3^−/− and control (WT) proplatelet-forming MKs. Scale bars are 50 μm. Data represent means ± standard deviations. Unpaired 2-tailed Student (I,K) or Welch (J) t test. ***P < .005.