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warning: country limit is at 12
wait, before someone asks what it means: country limit is the maximum number of regions that you can access simultaneously.
why is this limit not configurable?
a couple of clients may be sharing the same region code at once. i don’t know what it is.
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the original 2d pong game, often referred to as ping-pong, is a two-player video game where two players attempt to hit a tiny circular paddles and attempting to knock each other’s score into the other paddles. the difference between the two players is that one of them is white and the other one is black. any no score victory is called a “tie”. one of the two players can also “win by default” if they don’t score in either of the two games. in an 8 player game, the player with the highest score after three games is the winner. when there are four players, the player who scores the most points after 3 games is the winner of the game. if there are two players who achieve the same score, they play a series of matches in order to establish a higher score.
this article is published under the standard license to publish agreement. after one year the work will become freely available and the license terms will switch to a creative commons attribution-noncommercial-share alike 4.0 unported license.
in brief, transient transfection was performed using lipofectamine 2000 (invitrogen) according to the manufacturer’s instructions. to construct the flag-tagged plasmid containing wild type or mutated kras, we generated pcr products from plasmids containing wild type kras (forward, 5′- gcgatcggccgctagcgggatcaatcttcaaggg-3′; reverse, 5′- gcctcgagctagctatagacatctcatcgtcgga-3′) and mutated kras (forward, 5′-gcgatcggccgctagcgggatcaatcttcaagggcgacctccaagtgtgatcgatgagattttttctctttttttatttctggctgactccttttagctg-3′; reverse, 5′-gcctcgagctagcagtataaacgaggagcataggattctgtgttctgtgtcttcgattttatatgtatatctgactcgtcacatcac-3′). the pcr products were subcloned into the ecori-xhoi sites of the pcdna3.1 vector (invitrogen). twenty-four hours after transfection, cells were harvested for western blot analysis using anti-flag m2 antibody (sigma-aldrich).
briefly, monolayers of mcf-7 cells were washed and treated for 3 hr with 10% fbs medium containing vehicle (dmso) or ptk787 (100 nm). the cells were then stimulated for 2 h with 20 nm ngf. p-erk, and rpn2 were visualized by immunoblotting as described above. data are presented as means of three independent experiments. p-erk and rpn2 are normalized to total erk and actin, respectively. a two-tailed, two-sample equal variance student’s t-test was used to assess the significance of differences among groups (). protein phosphorylation was calculated relative to total protein content. all experiments were performed using minimum of three independent measurements.