GRK2 abundance is increased in NP-specific splenic ASCs in Zfp36l1^fl/flCD79a^Cre/+ mice. (A) An overlay of representative flow cytometry plots gated on CD138⁺NIP⁺IgG1⁺ cells from the spleens of Zfp36l1^fl/fl CD79a^+/+ and Zfp36l1^fl/fl CD79a^Cre/+ mice analyzed at days 3 and 5 after reimmunization. Antibody against GRK2 used in these experiments was from Cell Signaling Technology and was used in combination with the secondary Alexa Fluor 647–conjugated donkey anti-rabbit IgG antibody. (B) A plot showing geometric mean fluorescent intensities (gMFIs) for GRK2 signal in the population of CD138⁺NIP⁺IgG1⁺ cells. Each symbol represents data from an individual mouse, and lines represent means. Data are from independent experiments performed in confirmation of Fig. 4 F (n ≥ 4 per experiment; two-way ANOVA; the P value shown is for the main genotype effect). (C) A plot showing gMFIs for GRK2 signal in the population of CD138^highB220^low cells differentiated in vitro. GRK2 was detected with antibody from OriGene followed by the secondary Alexa Fluor 647–conjugated donkey anti-rabbit IgG antibody. Each symbol represents data from an individual mouse, and lines represent means. Data are from two independent experiments (n = 3 per experiment; two-way ANOVA; the P value shown is for the main genotype effect). (D) Representative Western blot of total protein extracts of CD138^highB220^low cells differentiated in vitro and sorted by a flow cytometer as described in Materials and methods. The upper panel shows the membrane incubated with antibody against GRK2, and the lower panels show the same membrane incubated with antibody against β-actin and against GAPDH. (E) A plot showing relative abundance of GRK2 as determined by a ratio of the signal for the band corresponding to GRK2 to the signal for β-actin (a comparable result was obtained with GRK2 signal normalized to that of GAPDH; data not shown). Each symbol represents data from an individual mouse, and lines represent means (n ≥ 2 per experiment; two-way ANOVA; the P value shown is for the main genotype effect). (F–H) Abundance of mRNA for the Itga4 (F), Itgb1 (G), and Itgb7 (H) genes in LPS-activated B cells from Zfp36l1^fl/fl CD79a^+/+ (closed circles) and Zfp36l1^fl/fl CD79a^Cre/+ (open squares) mice. Each symbol represents data from B cells of an individual mouse, and lines represent means. P values calculated with DESeq2 are adjusted by the Benjamini-Hochburg method. (I) Percentages of cells with a high integrin α4β7 surface abundance in the splenic populations of CD138⁺NIP⁺IgG1⁺ cells in Zfp36l1^fl/fl CD79a^+/+ (closed circles) and Zfp36l1^fl/fl CD79a^Cre/+ (open squares) mice at day 14 following primary immunization. Each symbol represents data from an individual mouse, and lines represent means. Data are representative of at least two independent experiments (n = 5; the P value is for a Student’s t test). (J and K) An overlay of representative flow cytometry plots showing surface expression of the integrin chains α4 (J) and β1 (K) on CD138⁺NIP⁺IgG1⁺ cells from the spleens of Zfp36l1^fl/fl CD79a^+/+ (shaded histogram) and Zfp36l1^fl/fl CD79a^Cre/+ (black line) mice analyzed 42 d after immunization.