Journal: The Journal of Biological Chemistry

Article Title: Heat-shock protein 90 (Hsp90) promotes opioid-induced anti-nociception by an ERK mitogen-activated protein kinase (MAPK) mechanism in mouse brain

doi: 10.1074/jbc.M116.769489

Figure Lengend Snippet: Hsp90 inhibition has multivariate effects on protein expression and signal transduction in vitro. Four different cell models of MOR signal transduction were treated with 1 μm 17-AAG or vehicle for 24 h, serum-starved (see “Experimental procedures”), and treated with vehicle, 10 μm DAMGO, or 10 μm LPA (a non-opioid GPCR endogenous agonist) for 10 min. Results were measured by Western blot, and analyzed as reported under “Experimental procedures.” The sample size reported in each experiment graph represents an independent technical replicate consisting of two or more averaged replicate wells counted as n = 1. All data are reported as the mean ± S.D. A, representative Western blots for each target are shown, with vehicle (Veh) or 17-AAG (AAG) treatment. The cell line from which each representative blot was taken is indicated below the target name. The numbers with arrows on the left side of each image represents the position of the nearest molecular weight markers. These images also indicate that GAPDH and tERK were not altered by 17-AAG treatment (data not shown). B, the expression levels of four separate signaling proteins normalized to GAPDH and vehicle control were analyzed in each cell line as labeled. *, ***, and ****, p < 0.05, 0.001, and 0.0001, respectively, versus same cell line vehicle control by unpaired 2-tailed t test. Only Hsp70 shows consistent regulation across cell lines with Hsp90 inhibition. C, signal transduction via ERK phosphorylation is reported for each cell line in a separate graph. The pERK signal was normalized to tERK and, in the case of CHO, was normalized further to MOR levels as they were decreased. The pERK/tERK signal was further normalized to the vehicle:vehicle group. * and **, p < 0.05 and 0.01 versus same pretreatment group (vehicle, 17-AAG) vehicle control; #, ##, ###, and ####, p < 0.05, 0.01, 0.001, and 0.0001, respectively, versus vehicle:vehicle group; both sets by two-way ANOVA with Fisher's least significant difference post-hoc test. Each line showed a different alteration in the ERK signaling pattern after Hsp90 inhibition, and both DAMGO and LPA showed similar behavior.

Article Snippet: The antibodies used were: Hsp70 (Cell Signaling 4872S, lot 4, rabbit, 1:1000), GAPDH (ThermoFisher MA5-15738, lot PI209504, mouse, 1:1000), STAT3 (Cell Signaling 9139S, lot 7, mouse, 1:1000), pERK (Cell Signaling 4370S, lot 12, rabbit, 1:1000), tERK (Cell Signaling 4696S, lot 16, mouse, 1:1000), pAkt (Cell Signaling 9018P, lot 3, rabbit, 1:1000), tAkt (Cell Signaling 2920S, lot 3, mouse, 1:1000), MOR (Abcam ab10275, lot GR81301-2, 1:500 for immunoprecipitation), β-arrestin2 (Abcam ab54790, rabbit, 1:500 for immunoprecipitation), β-arrestin2 (Cell Signaling 3857S, 2, rabbit, 1:1000 for Western blotting), HA tag for in vitro MOR (Cell Signaling 3724S, lot 3, rabbit, 1:1000), secondary GαM680 (LiCor 926-68020, lot C50721-02, goat, 1:10,000–1:20,000), and secondary GαR800 (LiCor 926-32211, lot {"type":"entrez-nucleotide","attrs":{"text":"C50602","term_id":"2387855","term_text":"C50602"}} C50602 –05, goat, 1:10,000–1:20,000).

Techniques: Inhibition, Expressing, Transduction, In Vitro, Western Blot, Molecular Weight, Control, Labeling, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Heat-shock protein 90 (Hsp90) promotes opioid-induced anti-nociception by an ERK mitogen-activated protein kinase (MAPK) mechanism in mouse brain

doi: 10.1074/jbc.M116.769489

Figure Lengend Snippet: Hsp90 inhibition induces protein expression and signaling changes in mouse brain. Mice were injected i.c.v. with vehicle or 0.5 nmol of 17-AAG, allowed to recover for 24 h, and then injected i.c.v. with vehicle (Veh) or 0.1 nmol of DAMGO for 10 min. Brains were extracted and analyzed by Western blot analysis as reported under “Experimental procedures.” All data are reported as the mean ± S.D. A, representative blots from each protein target are shown, with the treatment and molecular weight markers indicated as described in the legend for Fig. 1. PAG (B) and caudal brain stem (pons and medulla) (C) protein expression changes are indicated. The signal for each target is normalized to GAPDH and further normalized to the vehicle control for each. MOR and βarr2 in the PAG were measured by immunoprecipitation (see “Experimental procedures”). *, p < 0.05 versus same target vehicle control by unpaired 2-tailed t test. The reported sample size of the individual mice consists of two technical replicates performed by two different experimenters at different institutions. Only Hsp70 expression was altered by 17-AAG treatment in both brain regions, contrary to some of the results reported in the legend for Fig. 1. Importantly, GAPDH, tERK, and tAkt protein levels were not changed (see A; GAPDH and tAkt quantified data not shown). PAG (D) and caudal brain stem (E) ERK signaling changes are indicated. pERK was normalized to the tERK signal and further normalized to the vehicle:vehicle group. **, ***, and ****, p < 0.01, 0.001, and 0.0001, respectively, versus vehicle:vehicle group by Fisher's least significant difference post-hoc test. The reported sample size of individual mice for both D and E consisted of two technical replicates each performed by different experimenters at different institutions. Hsp90 inhibition increased the ERK signaling baseline, and stimulation above that baseline by DAMGO was completely lost in both brain regions. PAG (F) and caudal brain stem (G) Akt signaling changes are indicated. Akt was analyzed as described for ERK from the same samples and with the same technical replicates. * and **, p < 0.05 and 0.01 versus vehicle:vehicle group by Fisher's least significant difference post-hoc test. Hsp90 inhibition induced a very similar, albeit less robust pattern in Akt as in ERK. H, in a control experiment, ERK signaling was assessed in mice injected i.c.v. with the highest dose of 17-AAG tested in the dose response (5 nmol). PAG ERK signaling in these mice was assessed as described in D and E. *, p < 0.05 versus vehicle:vehicle group by Fisher's least significant difference post-hoc test. 5 nmol of 17-AAG induces the same ERK pattern as seen in D and E with no increased magnitude of the effect, suggesting that 0.5 nmol has a maximal effect on brain signaling, justifying the use of the lower dose. The reported sample size of individual mice consisted of one technical replicate.

Article Snippet: The antibodies used were: Hsp70 (Cell Signaling 4872S, lot 4, rabbit, 1:1000), GAPDH (ThermoFisher MA5-15738, lot PI209504, mouse, 1:1000), STAT3 (Cell Signaling 9139S, lot 7, mouse, 1:1000), pERK (Cell Signaling 4370S, lot 12, rabbit, 1:1000), tERK (Cell Signaling 4696S, lot 16, mouse, 1:1000), pAkt (Cell Signaling 9018P, lot 3, rabbit, 1:1000), tAkt (Cell Signaling 2920S, lot 3, mouse, 1:1000), MOR (Abcam ab10275, lot GR81301-2, 1:500 for immunoprecipitation), β-arrestin2 (Abcam ab54790, rabbit, 1:500 for immunoprecipitation), β-arrestin2 (Cell Signaling 3857S, 2, rabbit, 1:1000 for Western blotting), HA tag for in vitro MOR (Cell Signaling 3724S, lot 3, rabbit, 1:1000), secondary GαM680 (LiCor 926-68020, lot C50721-02, goat, 1:10,000–1:20,000), and secondary GαR800 (LiCor 926-32211, lot {"type":"entrez-nucleotide","attrs":{"text":"C50602","term_id":"2387855","term_text":"C50602"}} C50602 –05, goat, 1:10,000–1:20,000).

Techniques: Inhibition, Expressing, Injection, Western Blot, Molecular Weight, Control, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Chemokine CXCL1 Dimer Is a Potent Agonist for the CXCR2 Receptor

doi: 10.1074/jbc.m112.443762

Figure Lengend Snippet: FIGURE 3. Binding affinity and receptor activity of the CXCL1 trapped dimer. A, the binding affinities of CXCL1 WT (●) and trapped dimer (E) were calculated by measuring the inhibition of binding of 125I-CXCL1 to the CXCR2 receptor. B, Ca2 release activity of CXCL1 WT and trapped dimer are shown as a function of different doses. The calculated EC50 values indicate that the trapped dimer is a potent agonist. C, ERK phosphorylation activity of 10 nM CXCL1 WT and trapped dimer are shown at 1, 5, and 10 min. Results from densitometric analysis of Western blots (n 3) are expressed as mean S.D. (error bars) of the phosphorylated ERK normalized to total ERK for each sample. D, chemotaxis activity of WT and trapped dimer at 10 and 100 nM concentrations was measured using a Boyden Chamber-type assay. The data were collected in quadruplicate, and the results are expressed as mean S.D. (error bars) and are representative of three independent experiments.

Article Snippet: The rabbit anti-phospho-ERK (Thr-202/Tyr-204) and p44/p42 total ERK antibodies were from Cell Signaling, and APRIL 26, 2013 • VOLUME 288 • NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 12245 at U niv of St A ndrew s on M arch 4, 2015 http://w w w .jbc.org/ D ow nloaded from secondary goat anti-rabbit IgG antibody was from Santa Cruz Biotechnology.

Techniques: Binding Assay, Activity Assay, Inhibition, Phospho-proteomics, Western Blot, Chemotaxis Assay