AID interacts with the calcium and integrin binding protein 1.

<p><b>A</b>. myc-CIB1 co-IPs AID-EGFP but not EGFP only (left and right lanes, respectively). A myc-specific monoclonal antibody was used to precipitate complexes, and AID-GFP was detected with an α-GFP polyclonal antibody. <b>B</b>. AID-EGFP co-IPs myc-CIB1 in a DNase I- and RNase A-resistant manner. An α-GFP monoclonal antibody was used to precipitate AID-GFP, and myc-CIB1 was detected with an α-myc monoclonal antibody. <b>C</b>. AID-STZ pulls down endogenous CIB1 from HEK-293T cell extracts. IgG Sepharose was used to pull-down STZ complexes, and CIB1 was detected using an α-CIB1 polyclonal antibody. AID-STZ and GFP-STZ were detected with an α-strep antibody. A low level of non-specific background was observed in the vicinity of AID-STZ. For cell lysate (input) control blots, two panels are shown because GFP-STZ is expressed over 100-fold better than AID-STZ. A quantification of the input versus pull-down signal indicated that 1% of cellular CIB1 can be pulled-down with AID complexes when IgG sepharose beads are limiting.</p

CIB1 over-expression in DT40.

<p><b>A</b>. An IGC fluctuation analysis showing the percentage of surface Ig-positive cells in subclone cultures over-expressing human (h) or chicken (c) CIB1. Each X represents data from an individual subclone and the labeled horizontal bars report the medians for each data set. <b>B</b>. CIB1 over-expression confirmed by immunoblotting. Loading was controlled by stripping and re-probing the blot with an α-tubulin antibody.</p

AID localization in CIB^−/− DT40 cells.

<p><b>A</b>. AID-EGFP localization in CIB1^+/+ DT40. <b>B</b>. AID-EGFP localization in CIB1^−/− DT40. Images were taken using a 40× objective and the scale bars indicate 10 µm.</p

CIB1 is dispensable for immunoglobulin gene conversion in DT40.

<p><b>A</b>. Schematic showing the constructs used to replace exons 5 and 6 of <i>CIB1</i> with the indicated drug resistance cassettes. The positions of the allele-specific PCR primers for genotyping and the XhoI sites and the position of the 3′ external probe used for Southern blot analysis are shown. <b>B</b>. An agarose gel image showing the allele-specific PCR products from CIB1+/+, +/−, and −/− cell lines. <b>C</b>. An agarose gel image of CIB1-specific RT-PCR products from CIB1+/+, +/−, and −/− cell lines. AID-specific reactions were used to demonstrate the integrity of each cDNA preparation. <b>D</b>. An IGC fluctuation analysis showing the percentage of surface Ig-positive cells in subclone cultures of the indicated genotype. Each X represents data from an individual subclone and the labeled horizontal bars report the medians for each data set.</p

PAK1^−/− plantar perfusion and function in model of severe HLI in 6–8 week-old mice is similar to PAK1^+/+ mice.

<p>A) I/NI (Ischemic/Non-Ischemic) plantar perfusion ratio is comparable between PAK1^−/− and PAK1^+/+ mice as measured by laser Doppler imaging from day 0, immediately after HLI surgery, and throughout the 21 days after HLI surgery. B) Limb use score was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112239#s2" target="_blank">Materials and Methods</a> and is equivalent between groups, where a higher score is observed within the first 2 weeks after surgery and decreased significantly thereafter, and indicating recovery of limb function. C) Appearance scores of PAK1^+/+ and PAK1^−/− mice were not statistically significant. D) Laser Doppler images obtained immediately following HLI surgery and on day 21 reflect a lack of difference in perfusion between PAK1^+/+ (N = 8) and PAK^−/− (N = 11) mice.</p

Potential Compensation among Group I PAK Members in Hindlimb Ischemia and Wound Healing

<div><p>PAKs are serine/threonine kinases that regulate cytoskeletal dynamics and cell migration. PAK1 is activated by binding to the small EF hand protein, CIB1, or to the Rho GTPases Rac1 or Cdc42. The role of PAK1 in angiogenesis was established based only on <i>in vitro</i> studies and its role in angiogenesis <i>in vivo</i> has never been examined. Here we tested the hypothesis that PAK1 is an essential regulator of ischemic neovascularization (arteriogenesis and angiogenesis) and wound healing using a global PAK1 knockout mouse. Neovascularization was assessed using unilateral hindlimb ischemia. We found that plantar perfusion, limb use and appearance were not significantly different between 6–8 week old PAK1^−/− and PAK1^+/+ mice throughout the 21-day period following hindlimb ischemia; however a slightly delayed healing was observed in 16 week old PAK1^−/− mice. In addition, the wound healing rate, as assessed with an ear punch assay, was unchanged in PAK1^−/− mice. Surprisingly, however, we observed a notable increase in PAK2 expression and phosphorylation in ischemic gastrocnemius tissue from PAK1^−/− but not PAK1^+/+ mice. Furthermore, we observed higher levels of activated ERK2, but not AKT, in ischemic and non-ischemic muscle of PAK1^−/− mice upon hindlimb ischemic injury. A group I PAK inhibitor, IPA3, significantly inhibited endothelial cell sprouting from aortic rings in both PAK1^−/− and PAK1^+/+ mice, implying that PAK2 is a potential contributor to this process. Taken together, our data indicate that while PAK1 has the potential to contribute to neovascularization and wound healing, PAK2 may functionally compensate when PAK1 is deficient.</p></div

Unimpaired repair of cutaneous ear wounds in PAK1^−/− mice.

<p>A) Recovery of 2.0 mm ear punch wounds is represented by (Area _{day X/}Area _{day 0}) ×100. There was a notably larger but not statistically significant wound diameter in PAK1^−/− mice compared to PAK1^+/+ mice on day 7. B) The wound edge was stained with Mason's trichrome and shows a very similar wound closure between the two groups by day 28. n = 3 for both PAK1^+/+ and PAK1^−/− groups.</p

PAK1^−/− plantar perfusion and limb function following severe HLI in 16 week-old mice is slightly impaired compared to PAK1^+/+ mice, suggesting mild impairment in neovascularization in the absence of PAK1.

<p>A) I/NI foot perfusion ratio is similar between PAK1+/+ and PAK1−/− mice immediately following surgery, but starts to diverge by days 3 and 7 days (B–C). Worse use and appearance scores in PAK1^−/− mice on days 7 and 14 reflect the overall impaired limb function due to impaired neovascularization. D) Laser Doppler images obtained on day 0 pre- and post-surgery, and on day 21 showing similar perfusion between PAK1^−/− (N = 8) and PAK1^+/+ (N = 8) mice.</p

Increased protein expression and phosphorylation of PAK2 in ischemic gastrocnemius muscle in PAK1^−/− compared to PAK1^+/+ mice.

<p>A) Western blots showing upregulated PAK2 expression and a trend towards increased phospho-PAK2 in PAK1^−/− mice (lanes represent samples from 3 different mice). We did not observe expression of PAK3 in gastrocnemius tissue; however, abundant PAK3 expression is found in mouse brain tissue as can be seen from a positive control sample in lane 7 of the PAK3 blot. B–C) Densitometry analysis reveals a 2-fold increase in PAK2 expression in PAK1^−/− compared to PAK1^+/+ mice, normalized to GAPDH as a loading control, and a concomitant increase in phospho-PAK2 relative to total PAK2. *indicates p≤0.05 using Student's T-test, n = 6. D) Densitometric analysis of total PAK2 protein expression in <i>non-ischemic</i> muscle did not reveal a change in PAK1^+/+ versus PAK1^−/− mice.</p

CIB1 is dispensable for CSR in mice.

<p><b>A</b>. Relative levels of each antibody isotype in sera from CIB1^+/+ or CIB1^−/− mice as measured by ELISA. The CIB1^−/− data were normalized to the mean antibody levels in sera from wildtype (WT) littermates (arbitrarily set to 1 for comparison). Each X represents data from an independent animal and the horizontal bars and labels report the median values (n = 3 for CIB^+/+ and n = 6 for CIB^−/−). <b>B</b>. IgM to IgG1 CSR <i>ex vivo</i>. B-cells were isolated from the spleens of CIB1^+/+ or CIB1^−/− mice, cultured for 4 days in the presence of LPS and IL-4, and analyzed by α-IgG1-PE labeling and flow cytometry. Each X represents data from an independent animal and the horizontal bars and labels report the median values. <b>C</b>. Images of hematoxylin and eosin stained sections of spleen isolated from CIB1^+/+ and CIB1^−/− mice. Scale bars indicate 500 µm.</p